Methods of inhibiting tumor cell aggressiveness using the microenvironment of human embryonic stem cells

ABSTRACT

The invention provides compositions comprising one or more isolated factors from a microenvironment of human embryonic stem cells (hESCs), including, but not limited to, Lefty and inhibitors of Nodal. The invention also provides methods of utilizing factors derived from human embryonic stem cells (hESC) and their microenvironment to treat and prevent tumor formation and progression and to inhibit tumor cell aggressiveness. The invention further provides methods of inhibiting tumor cell growth and/or treating aggressive tumors in a mammal comprising administering to the mammal, having at least one tumor cell present in its body, an effective amount of an inhibitor of Nodal activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.11/829,070 filed on Jul. 26, 2007 now U.S. Pat. No. 7,666,423, whichclaims priority to U.S. Provisional Patent Application No. 60/941,343,filed Jun. 1, 2007 and U.S. Provisional Patent Application No.60/820,740 filed Jul. 28, 2006. All of the above applications areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to methods of using compounds produced byembryonic stem cells to treat and/or prevent the growth and/ordissemination of aggressive tumor cells in a patient. More specifically,the invention relates to the administration to the patient of inhibitorsof Nodal activity, including, but not limited to, those that areexclusively produced by human embryonic stem cells. The invention alsorelates to methods for detecting aggressive tumors in a patientcomprising detecting the presence of Nodal in the patient's cells.

BACKGROUND

Aggressive tumor cells share a number of characteristics with embryonicprogenitors. During vertebrate development, multipotent precursor cellsare gradually specified to particular fates through the autocrine orparacrine delivery of signaling molecules, and during cancerprogression, malignant cells similarly release and receive cues thatpromote tumor growth and metastasis. Aggressive tumor cells,particularly melanoma cells, display stem cell-like plasticity asdemonstrated by their molecular signature that signifies adedifferentiated, multipotent plastic phenotype (i.e. one that iscapable of responding to microenvironmental factors as well asinfluencing other cells via epigenetic mechanisms) (Bittner et al.,2000, Nature 406:536-540; Hendrix et al., 2003, Nat. Rev. Cancer3:411-421). Furthermore, aggressive melanoma cells are capable ofvasculogenic mimicry, i.e. they are able to form vasculogenic-likenetworks while simultaneously expressing genes associated with anendothelial cell type. (Seftor et. al., 2002, Crit. Rev. OncologyHematol. 44:17-27; Maniotis et. al., Am. J. Pathol. 155:739-752).

Previous studies capitalized on the similarities between cancer and stemcells by examining the ability of embryonic microenvironments tomodulate tumor cell behavior (Pierce et al., 1982, Cancer Res.42:1082-1087; Gerschenson et al., 1986, Proc. Natl. Acad. Sci. U.S.A83:7307-7310; Lee et al., 2005, Dev. Dyn. 233:1560-1570; Mintz et al.,1975, Proc. Natl. Acad. Sci. U.S.A 72:3585-3589). For example, Pierceand colleagues reported that neural stage mouse embryos regulateneuroblastoma cells, and that embryonic skin inhibits melanoma growth((Pierce et al., 1982, Cancer Res. 42:1082-1087; Gerschenson et al.,1986, Proc. Natl. Acad. Sci. U.S.A 83:7307-7310). Although studies havefocused on the role of embryonic signals in the regulation of tumorcells, few have utilized embryonic models as a tool to discovermolecular mechanisms by which cancer cells modulate theirmicroenvironment and the resulting reciprocal interactions.

One of the major factors contributing to the plasticity of stem cells isNodal. Nodal is a highly conserved morphogen belonging to thetransforming growth factor beta (TGFβ) super family (Schier et al.,2003, Annu. Rev. Cell Dev. Biol. 19:589-621). By acting as an organizingsignal before gastrulation, Nodal initiates embryonic axis formation,and previous studies demonstrated that the ectopic expression of Nodalinduces mesendodermal fates in ectopic positions (Whitman, 2001, Dev.Cell 1:605-617; Schier, 2003, Annu. Rev. Cell Dev. Biol. 19:589-621;Iannaccone et al., 1992, Dev. Dyn. 194:198-208; Smith, 1995, Curr. Opin.Cell Biol. 7:856-861; Zhou et al., 1993, Nature 361:543-547; Rebagliatiet al., 1998, Proc. Natl. Acad. Sci. U.S.A 95:9932-9937; Toyama et al.,1995, Development 121:383-391).

Activation of Nodal includes binding to the co-receptor Cripto andsubsequent phosphorylation of the type I and type II activin-like kinasereceptors (ALK). In turn, SMAD2 and SMAD3 are activated (Lee et. al.,2006, Nature Medicine 12:882-884). Furthermore, human embryonic stemcells express Nodal and secrete endogenous inhibitors of Nodal such asLefty A/B (Besser, D., 2004, J. Biol. Chem. 279:45076-45084). Lefty Aand Lefty B, human homologs to murine Lefty 2 and Lefty 1, respectively,are separated by approximately 50 kb on chromosome 1q42 and are 96%identical to each other (Kosaki et. al., 1999, Am. J. Hum. Genet.64:712-21). Lefty A and Lefty B are members of the TGFβ superfamily, andare considered one of the powerful inhibitors of Nodal.

SUMMARY OF THE INVENTION

The invention provides compositions comprising one or more isolatedfactors from a microenvironment of human embryonic stem cells (hESCs),including, but not limited to, Lefty and inhibitors of Nodal. Theinvention further provides an isolated Lefty protein produced byconditioning a matrix with human embryonic stem cells. The inventionfurther provides a protein comprising glycosylated Lefty, includingglycosylated Lefty isolated from the microenvironment of human embryonicstem cells, compositions thereof, and methods of inhibiting tumor cellgrowth in a mammal comprising administering to the mammal suchcompositions.

The invention also provides methods of utilizing factors derived fromhuman embryonic stem cells (hESC) and their microenvironment to treatand prevent tumor formation and progression and to inhibit tumor cellaggressiveness. The invention further provides methods of inhibitingtumor cell growth and/or treating aggressive tumors in a mammalcomprising administering to the mammal, having at least one tumor cellpresent in its body, an effective amount of an inhibitor of Nodalactivity, including, but not limited to, hESC-derived Lefty andsynthetic derivatives as discussed herein, glycosylated Lefty,recombinant Lefty, anti-Nodal antibodies, inhibitors of one or more ofactivin receptor-like proteins ALK 4, ALK 5, and/or ALK7, inhibitors ofCripto, anti-Nodal antisense moieties such as anti-Nodal Morpholinos,and Notch inhibitors including, but not limited to, Notch 4 inhibitorssuch as Notch 4 siRNA.

The invention also provides a method of inhibiting tumor cell growth ina mammal comprising administering to the mammal, having at least onetumor cell present in its body, an effective amount of a preconditionedmicroenvironment, which has been in contact with human embryonic stemcells.

The invention further provides methods for detecting aggressive tumors(including but not restricted to melanoma and breast carcinoma) in apatient comprising the steps of: obtaining a sample from a patient;assaying the sample for the presence of Nodal and the absence of a Nodalinhibitor (such as Lefty or modified Lefty or Lefty derivatives); anddetecting aggressive tumor cells if Nodal is present and Lefty is absentin the sample. The invention also provides methods of identifyingcompounds for treating aggressive tumors comprising providing aplurality of cells that express Nodal; assaying the cells in thepresence and absence of a candidate compound for activity of Nodal; andidentifying the compound as a compound for treating aggressive tumors ifthe Nodal activity is less in the presence of the candidate compoundthan in the absence of the candidate compound.

In addition, the invention provides methods for monitoring theeffectiveness of a pharmaceutical composition as an agent for treatingaggressive tumors in a patient, for detecting the presence of aggressivetumor cells, and for methods for detecting the presence of cells havinga dedifferentiated, multipotent plastic phenotype in a mammal

Specific embodiments of the invention will become evident from thefollowing more detailed description of certain preferred embodiments andthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an experimental methodology flow chart demonstrating theutilization of human embryonic stem cell microenvironments to inhibittumor cell aggressiveness.

FIG. 2 shows that the microenvironment of human embryonic stem cellsinduces melanoma cell spheroid formation. (A-G) Phase contrastmicroscopy showing the confluent growth of C8161 amelanotic, humanmetastatic cutaneous melanoma cells on 3-D Matrigel matrix (A), comparedwith the formation of colonies by H1 (B) and HSF-6 (C) human embryonicstem cells (hESCs) on 3-D Matrigel matrix; following removal of thehESCs from their 3-D matrix (leaving a denuded preconditioned matrix,CMTX, shown in inset), the C8161 tumor cells seeded onto the H1 (D,E)and HSF-6 (F) hESC preconditioned matrices, CMTX (Matrigel), now formspheroids (D-F) similar to hESC colonies. (Bar in “A” equals 200 μm). Incontrast, C8161 cells exposed to medium conditioned by H1 cells areunable to form spheroids (G).

FIG. 3 shows the epigenetic changes in human metastatic cutaneousmelanoma cells exposed to the microenvironment of human embryonic stemcells. (A) Western blot analysis of whole cell lysates (with an equalamount of protein loaded per sample), for a melanocyte marker, Melan-A,shows its absence in H1 hESCs on Matrigel and C8161 tumor cells onMatrigel; and the induction of Melan-A in C8161 cells exposed to the H1hESCs preconditioned matrix, CMTX (Matrigel), compared with Melan-A incontrol human epidermal melanocytes (HEMn) on Matrigel (upper panel).(B) Semi-quantitative RT-PCR analysis of Melan-A gene expression in HEMncultured on Matrigel compared to C8161 cells exposed to a HEMNpreconditioned matrix, CMTX (Matrigel), compared with C8161 cells onMatrigel. The CMTX lane serves as a control demonstrating the completeremoval of the HEMn cells from the preconditioned matrix prior toseeding the C8161 melanoma cells. GAPDH was used as a loading controlfor RNA (lower panel). (C) Semi-quantitative RT-PCR analysisdemonstrating that a collagen I 3-D matrix preconditioned by humanmelanocytes (HEMn18 or HEMn20 CMTX) does not change the expression ofthe genes tested. This indicates that the benign melanocytemicroenvironment does not epigenetically influence metastatic melanomacells to change their plastic, molecular phenotype.

FIG. 4 shows that the microenvironment of human embryonic stem cellsdecreases melanoma cell invasion and tumorigenesis. (A) Invasion ofC8161 cells following culture on unconditioned Matrigel (Control) orMatrigel preconditioned by either H1 or HSF-6 hESCs was calculated as apercentage of cells able to invade through a defined matrix (collagenIV, laminin, and gelatin)-coated membrane during a 24 hour period usingthe MICS (Membrane Invasion Culture System) assay. Bars represent themean, normalized, invasion indices±standard deviations. The valuesindicated by an asterisk (*) are significantly different from theinvasion index of control cells. (B) In vivo tumor formation in a mouseinjected with C8161 cells pre-exposed for 3 days to either a controlmatrix (Matrigel) or a matrix conditioned by hESCs (H9CMTX) (n=21).Values represent the median tumor volume (mm3)±interquartile range, andtumor volumes were significantly different at the time points indicatedby an asterisk (*) (P<0.05).

FIG. 5 shows differential expression of Nodal, Lefty, and Cripto inhESCs, aggressive tumor cells, and normal human cells. (A) Western blotanalyses of Nodal, Lefty and Cripto in: H1 and H9, human embryonic stemcell (hESC) lines; C8161, human metastatic melanoma cells; normal humanmelanocytes; MDA-MB-231, human metastatic breast carcinoma cells; Hs 578Bst normal human myoepithelial cells; and HMEpC normal human mammaryepithelial cells. Actin is used as a loading control. (B) Real TimeRT-PCR analysis of Lefty-B mRNA expression in H9, human embryonic stemcells (hESCs); C8161, human metastatic melanoma cells; normal humanmelanocytes; MDA-MB-231, human metastatic breast carcinoma cells; Hs 578Bst normal human myoepithelial cells; HMEpC normal human mammaryepithelial cells; GM00473 and GM00957A human amniotic fluid-derived stemcells; SC00125, human umbilical cord-derived stem cells; human adultmesenchymal stem cells (MSC); and HTR-8/SVneo, immortalized humancytotrophoblast cells. (C) Immunofluorescence localization of Cripto in100% of H9 hESCs and in small subpopulations of C8161 melanoma andMDA-MB-231 breast carcinoma cells. Bar equals 10 μm. (D) Western blotanalyses of Nodal, Lefty and Cripto in: H9, hESCs; GM00473 and GM00957Ahuman amniotic fluid-derived stem cells; SC00125, human umbilicalcord-derived stem cells; human adult mesenchymal stem cells (MSC); andHTR-8/SVneo, immortalized human cytotrophoblast cells. Actin is used asa loading control.

FIG. 6 shows patterns of Nodal Expression in Primary and MetastaticMelanoma Lesions. FIGS. 6 (A-F) show immunohistochemical analysis ofNodal staining in (A) normal skin, (B-D) a primary cutaneous melanomaand (E and F) cutaneous melanoma metastases. Arrows delineate (A) normalmelanocytes and (F) Nodal protein localized to melanoma cell membranes.(C) and (D) represent radial and vertical growth phases respectively.FIG. 6(A-D) are representative of 5 patient samples and FIG. 6(E-F) arerepresentative of 10 patient samples. Isotype controls are pictured inthe insets, and bars equal 50 μm.

FIG. 7 shows patterns of Nodal Expression in Breast Cancer Carcinoma.Immunohistochemical analysis of Nodal staining in normal breast tissue,ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC) andmetastatic IDC. Bars equal 100 μm.

FIG. 8 shows the distribution of Nodal and Lefty on hESCs cultured onMatrigel. Immunofluorescence localization of Lefty and Nodal in H9 hESCscultured on Matrigel and Western blot analysis of Lefty protein inmatrix conditioned by hESCs (H9 CMTX). Top panels representreconstructed confocal images depicting the cross-section of a hESCcolony with its underlying matrix. Dashed line designates thecell-matrix interface and the arrow points to the upper surface of thehESC colony. Corresponding images on the right illustrate Lefty andNodal at the cell surface (arrow) and the cell-matrix interface (dashedline). The large image is a 3-dimensional confocal projection of hESCcolonies stained with Lefty and Nodal (inset). Bar equals 25 μm.

FIG. 9 shows that the microenvironment of human embryonic stem cells(hESCs) leads to the reduction of Nodal expression and tumorgenicity inplastic metastatic melanoma and breast cancer cells exposed to theembryonic preconditioned matrix. (A) Western blot analyses demonstratingthat the microenvironments of hESCs reduced the expression of Nodalprotein in multipotent melanoma (C8161) and breast carcinoma(MDA-MB-231) cells. (B) Western blot analyses of Nodal protein in humanmetastatic melanoma cells (C8161) and human metastatic breast carcinomacells (MDA-MB-231) exposed for 3 days to either control (unconditioned)Matrigel or to Matrigel conditioned by hESCs (H9CMTX). Some cancer cellsexposed to H9CMTX were subsequently recovered on control (unconditioned)Matrigel for 2 or 7 days prior to Western blot analysis. Actin is usedas a loading control. (C) Real Time RT-PCR analysis of Nodal mRNA inhuman metastatic melanoma cells (C8161) and human metastatic breastcarcinoma cells (MDA-MB-231) exposed for 3 days to either control(unconditioned) Matrigel or to Matrigel conditioned by hESCs (H9CMTX).Some cancer cells exposed to H9CMTX were subsequently recovered oncontrol (unconditioned) Matrigel for 2 or 7 days prior to analysis. (D)Immunohistochemistry localization of Nodal in C8161 cells forming tumorsin nude mice at Day 19. Nodal staining is strongest in the Matrigelcontrol injected tumor cells vs. the diminished Nodal staining in C8161cells exposed to the H9CMTX (correlated with a lower tumor burden).(Bars equal 50 μm.) (E) Relative colony formation of C8161 andMDA-MB-231 cells cultured on soft agar for 14 days following 3 days ofexposure to either control (unconditioned) Matrigel or to H9CMTX. Assayswere conducted in the presence or absence of rNodal (100 ng/mL). Barsrepresent mean normalized colony formation±standard deviation. Thevalues indicated by an asterisk (*) are significantly different from thecolony forming ability of control cells and the values indicted by adouble asterisk (**) are significantly different from the colony formingability of control cells and H9CMTX treated cells (n=12, P<0.05). (F)Western blot analyses of Nodal protein in human metastatic melanomacells (C8161) and human metastatic breast carcinoma cells (MDA-MB-231)exposed for 3 days to either control (unconditioned) Matrigel or toMatrigel conditioned by normal melanocytes (melanocyte CMTX), normalmyoepithelial cells (Hs 578 Bst CMTX), amniotic fluid-derived stem cells(GM00473/GM00957A CMTX) or trophoblast cells (HTR-8/SVneo CMTX). Actinis used as a loading control. (G) Real Time RT-PCR analysis of NodalmRNA in human metastatic melanoma cells (C8161) exposed for 3 days toeither control (unconditioned) Matrigel or to Matrigel conditioned byamniotic fluid-derived stem cells (GM 00473/GM 00957A CMTX) ortrophoblast cells (HTR-8/SVneo CMTX). Gene levels were normalized using18s and bars represent mean gene expression normalized to H9 (A,B) orMatrigel (C,D) values.

FIG. 10 shows the role of hESC-derived Lefty in Nodal down-regulation.(A) An abundance of the Nodal inhibitor Lefty within the hESCconditioned matrices (CMTX). Lefty protein is absent in the C8161 cellswithin its own conditioned matrix (C8161+CMTX). (B) Immunofluorescencelocalization of FITC-conjugated anti Lefty Morpholinos (MO^(LEFTY)) inH9 hESC colonies on Matrigel. (C) Western blot analysis of Lefty proteinin H9 hESCs treated with either vehicle (Control), MO^(Control) (MOControl), or MO^(Lefty) (MO Lefty). Actin is used as a loading control.(D) Real Time RT-PCR analysis of Oct-3/4 and Nanog expression in H9hESCs treated with either vehicle (H9), MO^(CONTROL) (MO Control), orMO^(LEFTY) (MO Lefty). Gene levels were normalized using 18s and barsrepresent mean gene expression normalized to H9. (E) Real Time RT-PCRanalysis of Nodal mRNA expression in C8161 cells cultured for 3 days oncontrol (unconditioned) Matrigel, Matrigel conditioned by hESCs (H9CMTX)or Matrigel conditioned by hESCs in which Lefty protein expression wasknocked out with Lefty-specific Morpholinos (H9CMTX MO Lefty). (F)Western blot analysis of Nodal protein in human metastatic melanoma(C8161) and breast carcinoma (MDA-MB-231) cells exposed for 3 days toeither control (unconditioned) Matrigel or to Matrigel seeded with Leftyprotein purified from hESCs (H9-derived Lefty). MDA-MB-231 cells wereallowed to recover on fresh Matrigel for 2 days prior to analysis andActin is used as a loading control. (G) Relative colony formation ofC8161 and MDA-MB-231 cells cultured on soft agar for 14 days following 3days of exposure to either control Matrigel or Matrigel seeded withLefty purified from hESCs (Lefty), in the presence or absence of rNodal(100 ng/mL). Bars represent mean normalized colony formation±standarddeviation. The values indicated by an asterisk (*) are significantlydifferent from the colony forming ability of control cells (n=6,P<0.05).

FIG. 11 shows the down-regulation of Nodal by recombinant Lefty. Westernblot analysis of Nodal protein in C8161 cells exposed for 48 hrs tovarying concentrations (0-1000 ng/mL) of rLefty-B showing that theaddition of rLefty to C8161 cells reduces Nodal expression

FIG. 12 shows that hESC derived Lefty is glycosylated. Staining forglycoprotein and detection of Lefty-A and Lefty-B on a Western blotcontaining recombinant Lefty (rLefty)-B, rLefty-A and a lysate from H9human embryonic stem cells (hESCs) plus matrix conditioned by the H9hESCs for 3 days (CMTX). After SDS-polyacrylamide gel electrophoresis,the proteins were transblotted and stained for glycoproteins, identifiedby green bands (bottom). Lefty-A and -B were subsequently detected byWestern blot analysis (top). Arrows point to Lefty protein(s) on theWestern blot and to the identical locations on the image showing thestained glycoproteins.

FIG. 13 shows that Nodal inhibition and the microenvironment of hESCsabrogate tumorigenicity in vivo. (A) In vivo tumor formation in a mouseinjected with MDA-MB-231 cells pre-exposed for 3 days to either acontrol matrix (Matrigel) or a matrix conditioned by hESCs (H9CMTX)(n=10) (B) C81-61 cells, transfected with either an empty vector or aNodal expression construct (n=5), and (C) MDA-MB-231 cells treated witheither MO^(Control) or MO^(Nodal) (n=10). Values represent the meantumor volume (mm³)±standard error (A) or standard deviation (B,C), andtumor volumes were significantly different at the time points indicatedby an asterisk (*) (P<0.05). (D) The ratio of tumor cell proliferationto apoptosis for C8161 and MDA-MB-231 derived tumors, determined byimmunohistochemical staining for Ki67 and terminal deoxynucleotidyltransferase biotin-dUTP nick-end labeling (TUNEL). Prior to injectioninto a mouse, C8161 and MDA-MB-231 cells were cultured for 3 days oncontrol or hESC conditioned (H9CMTX) matrices, or treated withMO^(Nodal) to knock down Nodal expression. Bars represent meannormalized values±standard deviation, and values indicated by anasterisk (*) are significantly different from control values (P<0.05).(E) Immunohistochemical analysis of Ki67 expression (red/brown) andTUNEL staining in orthotopic melanoma (C8161) and breast carcinoma(MDA-MB-231) tumors. Prior to injection into a mouse, cells were treatedwith MO^(Nodal), exposed to H9 hESC CMTX, or left untreated (control).Proliferation is indicated by Ki67 staining and apoptotic nuclei weredetected with confocal microscopy as red staining localized to thenuclei of apoptotic C8161 or MDA-MB-231 cells. For the TUNEL analyses,cell nuclei are counterstained blue with DAPI. Bar equals 25 μm. (F) Invitro proliferation of C8161 and MDA-MB-231 cells treated with eitherMO^(Control) or MO^(Nodal). Values represent the mean cell count(×1000)±standard deviation 4 days after the plating of 15,000 cells. Anasterisk (*) indicates a significant difference between control andMO^(Nodal) treated cells (n=4, P<0.05).

FIG. 14 shows Nodal inhibition abrogates melanoma tumorigenicity. (A)Phase contrast microscopy of cells cultured for 7 days in a soft agarassay. The panels represent the colony-forming ability of poorlyaggressive C81-61 cells, aggressive C8161 cells, C8161 cells treatedwith MO_(Nodal) and C8161 cells treated with MO_(Nodal) and rescued withrecombinant Nodal (100 ng/mL). Bar equals 20 μm. (B) In vivo tumorformation in a mouse injected with C8161 cells treated with eitherMO_(Control) or MO_(Nodal). Values represent the median tumor volume(mm₃)±interquartile range, and the MO_(Control) and MO_(Nodal) tumorvolumes were significantly different at the time points indicated by anasterisk (*) (n=5, p<0.05). (C) Immunohistochemical analysis of Nodalstaining in an orthotopic tumor derived from C8161 cells treated withMO_(Nodal). The C8161 cells have begun to re-express Nodal by 17 dayspost-injection. Bar equals 50 μm. (MO_(Nodal)=antisense morpholino toNodal). (D) Acquisition of tumorigenic potential by C81-61 poorlyaggressive melanoma cells transfected with Nodal cDNA, compared withmock transfected C81-61 control: Values are reported as median tumorvolume after 38 days±standard deviation (*p<0.05; n=5/parameter).

FIG. 15 shows down-regulation of Nodal signaling results in acquisitionof a melanocyte-like phenotype and loss of the dedifferentiated, plasticphenotype. (A) Western blot analyses of Nodal, phosphorylated SMAD-2,total SMAD 2/3, and Actin in C8161 cells 48 hours after administrationof either vehicle or an ALK 4/5/7 inhibitor (SB431542, 1 μM, 10 μM).(A,B) All Nodal bands represent the pro-protein. (B) Western blotanalyses of Nodal, Tyrosinase and Actin in C8161 cells 24 hours afterthe administration of either vehicle or different concentrations of ALKinhibitor while (C) is a Western analyses for VE-Cadherin, Keratin 18and Actin in C8161 cells cultured on 3-D collagen I matrices for 6 daysin the presence of vehicle or different concentrations of ALK inhibitor.(D) Reduction in invasive ability and (E) abrogation of vasculogenicmimicry following down-regulation of Nodal with ALK 4/5/7 inhibitor.

FIG. 16 shows molecular cross-talk between Nodal and Notch. (A)Knockdown of Nodal expression in C8161 cells by Notch siRNAs,particularly Notch 4 siRNA. Western blot analysis of C8161 cells 72 hrsfollowing the administration of siRNA. Real-time RT-PCR and Western blotanalyses confirmed the silencing of each Notch at this time points. (B)Notch expression is relatively unaffected by knockdown of Nodal in C8161cells. Western blot analysis of Notch 1, 2 and 4 in C8161 cells treatedwith the Nodal inhibitor SB431542.

FIG. 17 shows that exposure of aggressive C8161 human melanoma cells tomatrices conditioned by hESCs induces an increase in the methylation ofspecific cytosine residues (CpG residues 6-17) in the first half of theCpG island in the Nodal promoter. The graphs depict the methylationstatus of the first half of the Nodal CpG island in aggressive C8161human cutaneous melanoma cells cultured on either Matrigel or onMatrigel conditioned by H9 hESCs. Each circle represents a CpGdinucleotide in the CpG island. Black and grey circles symbolizemethylated and unmethylated residues respectively. Each row representsan individual clone or allele. Although culture in the presence of ahESC microenvironment (H9CMTX) globally increases methylation by only6.8%, the shaded area experienced a 32% increase in methylation whencells were cultured on H9CMTX versus Matrigel alone.

DETAILED DESCRIPTION

In certain embodiments, the invention provides a composition comprisingone or more isolated factors from a microenvironment of human embryonicstem cells (hESCs). As used herein, “one or more isolated factors”refers to any one or any group of factors present in a microenvironmentof hESCs. The factors may be individually isolated, or isolated in amanner that provides a group of factors in combination. Alternatively,“one or more isolated factors” may refer to any one or a group offactors present on a defined matrix. As used herein, a“microenvironment” is an environment that comprises a basement membraneor other defined matrix that is in contact with embryonic stem cells,preferably human embryonic stem cells, and that is influenced by theembryonic stem cells. A “preconditioned” microenvironment is amicroenvironment that has been in contact with human embryonic stemcells under appropriate conditions as described herein, and describedfor example, in Postovit et al., 2006, Stem Cells 24:501-505 andillustrated in FIG. 1 herein. FIG. 1 herein illustrates utilization ofhuman embryonic stem cell microenvironments to inhibit tumor cellaggressiveness.

In one embodiment, the isolated factor(s) from a microenvironment ofhESCs inhibit Nodal. As described herein, aggressive tumor cells expressNodal, and Nodal is essential for plasticity, tumorgenicity andaggressiveness. Therefore, inhibiting Nodal provides an excellentapproach for treating and preventing aggressive tumors. As used herein,the terms “aggressive tumor” and “aggressive cancer,” which include“aggressive melanoma” and “aggressive breast carcinoma” refer to amalignant cell that has neoplastic growth with or without metastaticinvolvement. In a non-limiting example, an aggressive tumor may refer toa malignant cell that has a transdifferentiated phenotype characterizedby the aberrant expression of genes normally restricted to other celllineages, concomitant with the loss of lineage-specific factors. Forexample, aggressive melanoma cells possess keratin-positive,intermediate filaments indicative of epithelial cells, and theyaberrantly express genes, including VE-Cadherin, normally associatedwith endothelial cells. Furthermore, the expression of melanocytespecific markers, such as Tyrosinase, is dramatically reduced, andsometimes absent, in aggressive melanoma cells. Tyrosinase catalyses theconversion of tyrosine to the pigment melanin, and is reduced by morethan 35-fold in aggressive melanomas as compared to their poorlyaggressive counterparts. Aggressive tumor cells also have the ability toexpress multiple stem cell markers, suggestive of a multipotent,dedifferentiated phenotype. These aggressive tumor cells are also highlymetastatic.

In one embodiment of the invention, the factor from a microenvironmentof hESCs is Lefty. As noted herein, Lefty, including hESC-derived Lefty,is an inhibitor of Nodal. As used herein, the terms “Lefty A/B” and“Lefty” are interchangeable and refer to either Lefty A or Lefty B, orboth Lefty A and Lefty B in combination. In one embodiment, Lefty,isolated from a microenvironment, may be substantially pure. In anotherembodiment, Lefty may be present in combination with other hESC factors.

In another embodiment, the invention provides an isolated Lefty proteinproduced by conditioning a matrix with human embryonic stem cells. Asused herein, “conditioning a matrix” refers to preparing apreconditioned microenvironment as defined herein. In certainembodiments, the matrix is conditioned with hESCs from 0 to 10 days orany range in between, including, but not limited to, from 0.5 to 10days, from 2 to 8 days, from 3 to 6 days, from 3 to 5 days, from 3 to 4days, or for 1, 2, 3, 4, or 5 days. Lefty may be isolated from thematrix by any method known to one of skill in the art, including throughuse of anti-Lefty antibodies.

In one embodiment, the invention provides a protein comprisingglycosylated Lefty. In this embodiment, Lefty may be glycosylated tovarying degrees, and may comprise one or more N- and/or O-linkedglycosylation sites, or a combination thereof. In one embodiment, theglycosylated Lefty is characterized in that more than 0%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or 95% of the possible N- and/or O-glycosylation sites areglycosylated. In another embodiment, the glycosylated Lefty ischaracterized in that less than 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of thepossible N- and/or O-glycosylation sites are glycosylated. In anotherembodiment, the glycosylated Lefty is characterized in that thepercentage of possible N- and/or O-glycosylation sites that areglycosylated is based on a combination of the “more than” and “lessthan” percentages recited above. Thus, in one non-limiting example, theglycosylated Lefty is characterized in that more than 30% and less than70% of the possible N- and/or O-glycosylation sites are glycosylated. Inanother embodiment, 100% of the possible N- and/or O-glycosylation sitesare glycosylated.

In one embodiment, the glycosylated Lefty is glycosylated tosubstantially the same extent as Lefty derived from hESCs.

Glycosylated Lefty may be prepared by any method, including byrecombinant methods (see, e.g. Sambrook et al., 2001, Molecular Cloning:A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). In one embodiment, glycosylated Lefty is preparedrecombinantly in Chinese Hamster Ovary (CHO) cells. Alternatively,glycosylated Lefty may be prepared by chemical synthesis methods (suchas solid phase peptide synthesis) using techniques known in the art suchas those set forth by Merrifield et al., 1963, J. Am. Chem. Soc.85:2149; Houghten et al., 1985, Proc Natl Acad. Sci. USA 82:5132; andStewart and Young, Solid Phase Peptide Synthesis (Pierce Chemical Co.1984), or by a combination of synthetic and recombinant techniques.Glycosylated Lefty may also be prepared by isolation from hESCs,including by isolation from the microenvironment of hESCs.

Included within the scope of the invention are fragments or derivativesof Lefty or glycosylated Lefty. As used herein, “fragment” means anyportion of the full length Lefty sequence having an activity of the fulllength protein, including, but not limited to, the ability to inhibitNodal. Included in the scope of “fragments” are naturally occurringenzymatic cleavage products. Included in the scope of the term“derivatives” are derivatives of full length Lefty as well as fragmentsthereof. As used herein, “derivative” or “derivatives” includesvariations of Lefty having one or more amino acid residues which havebeen added, deleted, inserted or substituted, where the resultingpolypeptide has an activity of Lefty, including, but not limited to, theability to inhibit Nodal. As used herein, “derivatives” also includeschemical derivatives of Lefty and variations thereof. It will beunderstood to one of skill in the art that these variations may occur inany combination.

Chemically modified derivatives of glycosylated Lefty may be prepared byone skilled in the art, in view of the disclosures described herein.Glycosylated Lefty derivatives are modified in a manner that isdifferent—either in the type or location of the molecules naturallyattached to the polypeptide. Derivatives may include molecules formed bythe deletion of one or more naturally-attached chemical group, or theymay be modified by the covalent attachment of one or more polymers. Forexample, the polymer selected is typically water-soluble so that theprotein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. Included within thescope of suitable polymers is a mixture of polymers. Preferably, fortherapeutic use of the end-product preparation, the polymer will bepharmaceutically acceptable.

The polymers each may be of any molecular weight and may be branched orunbranched. The polymers each typically have an average molecular weightof between about 2 kDa to about 100 kDa (the term “about” indicatingthat in preparations of a water-soluble polymer, some molecules willweigh more, some less, than the stated molecular weight). The averagemolecular weight of each polymer is preferably between about 5 kDa andabout 50 kDa, more preferably between about 12 kDa and about 40 kDa andmost preferably between about 20 kDa and about 35 kDa.

Suitable water-soluble polymers or mixtures thereof include, but are notlimited to, N-linked or O-linked carbohydrates, sugars, phosphates,polyethylene glycol (PEG) (including the forms of PEG that have beenused to derivatize proteins, including mono-(C.sub.1-C.sub.10), alkoxy-,or aryloxy-polyethylene glycol), monomethoxy-polyethylene glycol,dextran (such as low molecular weight dextran of, for example, about 6kD), cellulose, or other carbohydrate based polymers, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols(e.g., glycerol), and polyvinyl alcohol. Also encompassed by the presentinvention are bifunctional crosslinking molecules which may be used toprepare covalently attached glycosylated Lefty polypeptide multimers.

In general, chemical derivatization may be performed under any suitablecondition used to react a protein with an activated polymer molecule.The optimal reaction conditions will be determined based on knownparameters and the desired result. For example, the larger the ratio ofpolymer molecules to protein, the greater the percentage of attachedpolymer molecule. In one embodiment, the glycosylated Lefty derivativemay have a single polymer molecule moiety at the amino-terminus. See,e.g., U.S. Pat. No. 5,234,784.

The pegylation of a polypeptide may be specifically carried out usingany of the pegylation reactions known in the art. Such reactions aredescribed, for example, in the following references: Francis et al.,1992, Focus on Growth Factors 3:4-10; European Patent Nos. 0154316 and0401384; and U.S. Pat. No. 4,179,337.

In another embodiment, glycosylated Lefty polypeptides may be chemicallycoupled to biotin. The biotin/glycosylated Lefty polypeptide moleculesare then allowed to bind to avidin, resulting in tetravalentavidin/biotin/glycosylated Lefty polypeptide molecules. GlycosylatedLefty polypeptides may also be covalently coupled to dinitrophenol (DNP)or trinitrophenol (TNP) and the resulting conjugates precipitated withanti-DNP or anti-TNP-IgM to form decameric conjugates with a valency of10.

Generally, conditions that may be alleviated or modulated by theadministration of the present glycosylated Lefty derivatives includethose described herein for glycosylated Lefty. However, the glycosylatedLefty derivatives disclosed herein may have additional activities,enhanced or reduced biological activity, or other characteristics, suchas increased or decreased half-life, as compared to the non-derivatizedmolecules.

In a further embodiment, the present invention provides a compositioncomprising a glycosylated Lefty. Compositions may be formulated as knownto one of skill in the art or as described herein. In anotherembodiment, the present invention provides methods of inhibiting tumorcell growth in a mammal comprising administering to the mammal acomposition comprising a glycosylated Lefty at a physiologicallyacceptable dosage. Such a composition may be administered in aneffective or therapeutically effective amount. As used herein,“effective amount” and “therapeutically effective amount” are usedinterchangeably.

By mammal it is meant humans, companion animals such as cats and dogs,primates such as monkeys and chimpanzees, and livestock animals such ashorses, cows, pigs, and sheep, or any patient in need of, or that willbenefit from, administration of any of the methods or compounds orcompositions of the invention. The term “patient” as used hereinincludes human and animal subjects.

In one embodiment, the invention comprises methods of inhibiting tumorcell growth in a mammal comprising administering to the mammal acomposition comprising a glycosylated Lefty at a dosage between 0.01 and500 ng/mL, between 0.01 and 200 ng/mL, between 0.1 and 200 ng/mL,between 0.1 and 100 ng/mL, between 1 and 100 ng/mL, between 10 and 100ng/mL, between 10 and 75 ng/mL, between 20 and 75 ng/mL, between 20 and50 ng/mL, between 25 and 50 ng/mL, or between 30 and 40 ng/mL. Inanother embodiment, the invention comprises methods of inhibiting tumorcell growth in a mammal comprising administering to the mammal acomposition comprising a glycosylated Lefty at a dosage of about 1, 5,10, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, or 500 ng/mL. As used inthis context, “about” means within 0, 1, 2, or 3 ng/mL of the recitedconcentration.

In certain embodiments, the invention provides methods of using one ormore factors from a microenvironment of human embryonic stem cells toinhibit tumor cell aggressiveness. In one embodiment, the factor(s) isan inhibitor of Nodal, including, but not limited to Lefty andglycosylated Lefty.

In one embodiment of these methods, the factor(s) inhibiting tumor cellaggressiveness do so by increasing apoptosis. As used herein,“apoptosis” refers to the physiologic process of programmed cell deathwhich normally occurs during embryonic development and duringmaintenance of tissue homeostasis. In a further embodiment, thefactor(s) inhibiting tumor cell aggressiveness do so by decreasing cellproliferation. Cell proliferation is defined as the increase in numberof cells resulting from completion of the cell cycle, as contrast togrowth, which is the increase in the individual cell mass. In a furtherembodiment, the factor(s) inhibiting tumor cell aggressiveness do so byboth increasing apoptosis and by decreasing cell proliferation and/or bydecreasing the tumor cell proliferation-to-apoptosis ratio.

In another embodiment, the invention provides a method of inhibitingtumor cell growth in a mammal comprising administering to the mammal,having at least one tumor cell present in its body, an effective amountof a preconditioned microenvironment, which has been in contact withhuman embryonic stem cells.

In further embodiments of the invention, Nodal and/or Lefty are used asbiomarkers for aggressive tumor cell aggressiveness and for prognostic,diagnostic and clinical diagnoses for aggressive carcinoma including,but not limited to, melanoma and breast cancer. In certain embodiments,the invention provides methods for detecting aggressive tumors(including but not restricted to melanoma or breast cancer) in a patientcomprising the steps of: obtaining a sample from a patient; assaying thesample for the presence of Nodal and Lefty; and detecting aggressivetumors if Nodal is present and Lefty is absent in the sample. As used inthis context, “a sample” includes, but is not limited to, tumor cells,tissue samples, and bodily fluids as defined herein. In a non-limitingexample, the sample can be serum.

In certain embodiments, the presence of Nodal can be detected byassaying for the Nodal gene or gene product. For example, a nucleic acidbased assay or a protein based assay can be used to detect the presenceof Nodal in a tumor sample. Exemplary assays that can be used to detectNodal include those described herein. The presence of Lefty can besimilarly detected. Those of skill in the art readily recognize thatother assays can be designed following conventional methods asdescribed, for example, in Sambrook et al., 2001, Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

In another embodiment, compounds for treating aggressive tumors may beidentified by providing a plurality of cells that express Nodal,assaying the cells for Nodal activity in the presence and absence of acandidate compound, and identifying the compound as a compound suitablefor treating aggressive tumors if the Nodal activity is less in thepresence of the compound than in the absence of the candidate compound.As used in this context, “Nodal activity” refers Nodal expression and/orto any of the activities recited herein, including maintaining tumorcell plasticity, tumorgenicity and aggressiveness.

In another method of the invention, the effectiveness of apharmaceutical composition as an agent for treating aggressive tumors ina patient may be monitored. The method comprises obtaining a firstsample from a patient; assaying the first sample for the presence ofNodal; administering an amount a pharmaceutical composition to thepatient; assaying subsequently-collected biological samples from thepatient for the presence of Nodal; and comparing the amount of Nodaldetected in the first sample with the amount of Nodal detected in thesubsequent samples, wherein the effectiveness of the pharmaceuticalcomposition is monitored by detecting changes in the amount of Nodal inthe subsequently-collected samples compared with the first sample. Asused in this context, “a sample” or “biological sample” includes, but isnot limited to, tumor cells, tissue samples, and bodily fluids asdefined herein. In a non-limiting example, the sample can be serum.

In another method of the invention, the presence of aggressive tumorcells in a mammal may be detected by obtaining a sample of tumor cellsfrom a patient; conducting a sequence based methylation analysis of theNodal CpG island in the tumor cells; comparing the degree of methylationin the CpG island of Nodal in the tumor cells to that of non-aggressiveor non-tumor cells; and correlating hypermethylation of Nodal with thepresence of aggressive tumor cells. The sequence based methylationanalysis may be based on the entirety of the CpG island or on asubsection thereof. In a further method of the invention the presence ofcells having a dedifferentiated, multipotent plastic phenotype in amammal may be detected by obtaining a sample from a mammal; assaying thesample for the presence of Nodal; and correlating the presence of Nodalwith the presence cells having a dedifferentiated, multipotent plasticphenotype. The sample may be a bodily fluid. Bodily fluids include, butare not limited to, whole blood, blood plasma, blood serum, urine,semen, saliva, lymph fluid, meningal fluid, amniotic fluid, glandularfluid, sputum and cerebrospinal fluid. Bodily fluid also includesexperimentally separated fractions of all of the preceding and solutionsor mixtures containing homogenized solid material, such tissues andbiopsy samples. These methods may be used as a prognostic or diagnosticassay for aggressive cancer or susceptibility to aggressive cancer,including, but not limited to, melanoma and breast cancer.

In other embodiments, the invention provides methods of inhibiting tumorcell growth in a mammal comprising administering to the mammal, havingat least one tumor cell present in its body, an effective amount of aninhibitor of Nodal activity.

The invention also provides methods of treating aggressive tumors in amammal comprising administering to the mammal, having at least one tumorcell present in its body, an effective amount of an inhibitor of Nodalactivity. As used herein, the phrase “treating aggressive tumors” refersto a method comprising administering a Nodal inhibitor to a mammal inneed thereof, wherein the Nodal inhibitor prevents aggressive tumor cellgrowth, and/or prevents aggressive tumor cell metastasis in the mammal.

In one embodiment, the invention provides methods of inhibiting tumorcell growth and/or treating aggressive tumors comprising contacting thetumor cell with a microenvironment that comprises human embryonic stemcells or a microenvironment that has been preconditioned by humanembryonic stem cells (“CMTX”). In certain embodiments, the basementmembrane matrix can be Matrigel™. There is variability between lots ofMatrigel basement membrane matrix, which can impact the preparation ofthe preconditioned media. More specifically, occasional lots of Matrigelwill not produce a preconditioned microenvironment that has the tumorinhibiting properties of the invention. In such situations, an alternatelot can be used. One of skill in the art will understand that othermatrices may be used.

As used herein, an “inhibitor” can be any chemical compound, nucleicacid molecule, endogenous protein such as Lefty A/B, peptide orpolypeptide such as an antibody against Nodal that can reduce Nodalactivity or interfere with expression of a Nodal gene. Included withinthe scope of the term “inhibitor” is any combination of two or more suchinhibitors administered concurrently or separately and in any order. ANodal inhibitor can inhibit the activity of a Nodal protein eitherdirectly or indirectly. Direct inhibition can be accomplished, forexample, by binding to a Nodal protein and thereby preventing the Nodalprotein from binding an intended target, such as a receptor. Indirectinhibition can be accomplished, for example, by binding to a Nodalprotein's intended target, such as a receptor or binding partner,thereby blocking or reducing activity of the Nodal protein. Furthermore,a Nodal inhibitor can inhibit a Nodal gene by reducing or inhibitingexpression of the gene, inter alia by interfering with gene expression(transcription, processing, translation, post-translationalmodification), for example, by interfering with the Nodal mRNA andblocking translation of the Nodal gene product or by post-translationalmodification of the Nodal gene product, or by causing changes inintracellular localization.

A Nodal inhibitor can also be an endogenously produced protein,including but not restricted to, Lefty A/B derived from themicroenvironment of human embryonic stem cells. For example, Lefty A/Bis produced in human embryonic stem cells and is secreted into themicroenvironment surrounding the cells. Lefty A/B can be isolated fromthe microenvironment. Alternatively, Lefty A/B can be isolated from thehuman embryonic stem cells directly (i.e. before it is secreted into themicroenvironment). In another embodiment, a Nodal inhibitor within thescope of the invention is recombinant Lefty A/B (rLefty) that may beprepared by any conventional methods known in the art. Lefty A/B may beglycosylated or non-glycosylated. In certain embodiments, Nodalinhibitors in accordance with the invention are glycosylated Lefty A/Bproduced by hESCs. In other embodiments, glycosylated Lefty A/B may beprepared by using CHO (Chinese Hamster Ovary) cells. In some instances,glycosylated Lefty A/B may be a more potent inhibitor of Nodal than itsnon-glycosylated or recombinant counterpart, and may therefore beadministered in therapeutic applications at a lower dose.

In other embodiments, Nodal inhibitors are molecules which interferewith Nodal signaling, such as activin-like kinase (ALK) inhibitors. Forexample, Nodal propagates its signal by binding to heterodimericcomplexes between type I (ALK 4/5/7) and type II (ActRIIB) activin-likekinase receptors. Assembly of the complex causes phosphorylation andactivation of ALK 4/5/7 by ActRIIB, which is followed by ALK 4/5/7mediated phosphyorylation of Smad-2/3 Inhibitors of ALK 4, ALK 5, and/orALK7 are included within the scope of the invention; as describedherein, ALK 4/5/7 inhibitors can abrogate Nodal expression. In oneembodiment, the ALK inhibitor is SB431542 (Sigma, St. Louis, Mo.).

In one embodiment, an inhibitor can be, for example, a small moleculeinhibitor, an antibody, a nucleic acid such as an antisenseoligonucleotide, a short interfering RNA (siRNA) molecule, or a shorthairpin RNA (shRNA) molecule. In addition, such inhibitors can bespecifically designed using the methods described herein or usingmethods known in the art.

In certain embodiments, an antisense oligonucleotide is complementary toat least a portion of a Nodal gene, so long as hybridization of theantisense oligonucleotide inhibits Nodal activity. The term“oligonucleotide” as used herein includes naturally occurring, andmodified nucleotides linked together by naturally occurring, and/ornon-naturally occurring oligonucleotide linkages. Oligonucleotides are apolynucleotide subset generally comprising no more than 200 nucleotides.In certain embodiments, oligonucleotides are 10 to 60 nucleotides inlength. In certain embodiments, oligonucleotides are 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 to 40 bases in length.Oligonucleotides are single stranded, e.g. for use in the constructionof a gene mutant using site directed mutagenesis techniques.

The oligonucleotides of the invention may also comprise nucleotideanalogs that may be better suited as therapeutic or experimentalreagents. An example of an oligonucleotide analogue is a peptide nucleicacid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in theDNA (or RNA), is replaced with a polyamide backbone which is similar tothat found in peptides (P. E. Nielsen, et al Science, 1991, 254, 1497).PNA analogues have been shown to be resistant to degradation by enzymesand to have extended lives in vivo and in vitro. PNAs also bind strongerto a complimentary DNA sequence due to the lack of charge repulsionbetween the PNA strand and the DNA strand. Other oligonucleotides maycontain nucleotides containing polymer backbones, cyclic backbones, oracyclic backbones. For example, the nucleotides may have morpholinobackbone structures (U.S. Pat. No. 5,034,506). Oligonucleotides may alsocontain groups such as reporter groups, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide.Oligonucleotides may also have sugar mimetics.

The antisense nucleic acid molecules may be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. The antisense nucleic acid molecules of the invention or a fragmentthereof, may be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed with mRNA or the native gene e.g.phosphorothioate derivatives and acridine substituted nucleotides. Theantisense sequences may be produced biologically using an expressionvector introduced into cells in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense sequences are producedunder the control of a high efficiency regulatory region, the activityof which may be determined by the cell type into which the vector isintroduced.

In one embodiment, the Nodal inhibitors of the invention are anti-NodalMorpholinos.

In one embodiment, certain inhibitors provided by the invention arespecies of short interfering RNA (siRNA). The term “short interferingRNA” or “siRNA” as used herein refers to a double stranded nucleic acidmolecule capable of RNA interference or “RNAi”, as disclosed, forexample, in Bass, 2001, Nature 411: 428-429; Elbashir et al., 2001,Nature 411: 494-498; and Kreutzer et al., International PCT PublicationNo. WO 00/44895; Zernicka-Goetz et al., International PCT PublicationNo. WO 01/36646; Fire, International PCT Publication No. WO 99/32619;Plaetinck et al., International PCT Publication No. WO 00/01846; Melloand Fire, International PCT Publication No. WO 01/29058;Deschamps-Depaillette, International PCT Publication No. WO 99/07409;and Li et al., International PCT Publication No. WO 00/44914. As usedherein, siRNA molecules need not be limited to those moleculescontaining only RNA, but further encompasses chemically modifiednucleotides and non-nucleotides having RNAi capacity or activity.Specific siRNA molecules that inhibit Nodal activity can be designedusing methods known to those of skill in the art or commerciallyavailable technology (such as technology provided by Dharmacon Research,Lafayette, Colo.).

In another embodiment, the Nodal inhibitors of the invention include anychemical compounds, nucleic acids, proteins, peptides, polypeptides,antibodies, or other molecules that inhibit Notch. In certainembodiments, the Nodal inhibitors are Notch4 inhibitors. In certainembodiments the Nodal inhibitors are Notch siRNAs. In certainembodiments, the Nodal inhibitors are Notch4 siRNAs.

In certain embodiments, the invention provides antibodies orimmunologically functional fragments thereof that selectively bind toNodal and methods for selectively inhibiting or interfering with theactivity of Nodal proteins. Standard methods for preparation ofmonoclonal and polyclonal antibodies and immunologically activefragments thereof are well known in the art, for example as described inHarlow and Lane (1988, ANTIBODIES: A LABORATORY MANUAL, Cold SpringHarbor Laboratory Press: New York). Methods for generating antibodyfragments, particularly Fab fragments and other fragments that retainepitope-binding ability and specificity are also well known, as arefully human antibodies and chimeric antibodies, including “humanized”antibodies. “Humanized” antibodies include, for example, antibodiesgenerated in mice that are “humanized” to reduce negative immune effectsthat can occur during administration to human subjects by replacingcertain portions of the mouse antibody with portions of humanantibodies. Thus, the invention encompasses use of antibody inhibitorsof Nodal that include, but are not limited to, single chain antibodies,single chain Fv antibodies, F(ab) antibodies, F(ab)′ antibodies and(Fab′)₂ antibodies, chimeric antibodies in which one or more regionshave been replaced by homologous human or non-human portions, and fullyhuman antibodies. Single chain antibodies are discussed in detail inInternational Patent Application Publication No. WO 88/01649 and U.S.Pat. Nos. 4,946,778 and 5,260,203. Such inhibitors can be delivered, forexample, via a penetratin tag (HIV or antennaepedia) or by recombinantmeans (e.g. encoded by a polynucleotide introduced into a cell in aviral vector).

In preferred embodiments, methods of the invention comprise the step ofadministering a pharmaceutical composition comprising an effectiveamount of one or a plurality of Nodal inhibitors together with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant, wherein the pharmaceutical composition iscapable of inducing a desired therapeutic effect when properlyadministered to a patient. Preferably, acceptable formulation materialsare nontoxic to recipients at the dosages and concentrations employed.

The expression “effective amount” in reference to a pharmaceuticalcomposition comprising one or a plurality of Nodal inhibitors isunderstood to mean, according to the invention, an amount of the saidpharmaceutical composition that is capable of preventing or reducinggrowth of aggressive melanoma cells. For example, a pharmaceuticalcomposition is therapeutically effective where a patient who hasaggressive melanoma has a reduced number of melanoma cells and/orreduced metastases of melanoma cells after treatment with thepharmaceutical composition compared with prior to said treatment. Apharmaceutical composition administered to a patient is alsotherapeutically effective where metastases of melanoma cells areprevented from occurring in a patient who has melanoma, has a history ofmelanoma (e.g. patient is in remission), or who is considered likely topresent with melanoma (e.g. has a genetic disposition favoring onset ofmelanoma).

In certain embodiments, a pharmaceutical composition useful in themethods of the invention may contain formulation materials formodifying, maintaining or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption or penetration of the composition.In such embodiments, suitable formulation materials include, but are notlimited to, amino acids (such as glycine, glutamine, asparagine,arginine or lysine); antimicrobials; antioxidants (such as ascorbicacid, sodium sulfite or sodium hydrogen-sulfite); buffers (such asborate, bicarbonate, Tris-HCl, citrates, phosphates or other organicacids); bulking agents (such as mannitol or glycine); chelating agents(such as ethylenediamine tetraacetic acid (EDTA)); complexing agents(such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. SeeREMINGTON'S PHARMACEUTICAL SCIENCES, 18^(th) Edition, (A. R. Gennaro,ed.), 1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theNodal inhibitors.

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition may be either aqueous or non-aqueous innature. For example, a suitable vehicle or carrier may be water forinjection, physiological saline solution or artificial cerebrospinalfluid, possibly supplemented with other materials common in compositionsfor parenteral administration. Neutral buffered saline or saline mixedwith serum albumin are further exemplary vehicles. In preferredembodiments, pharmaceutical compositions of the present inventioncomprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH4.0-5.5, and may further include sorbitol, sucrose, Tween-20 and/or asuitable substitute therefor. In certain embodiments of the invention,Nodal inhibitor compositions may be prepared for storage by mixing theselected composition having the desired degree of purity with optionalformulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in theform of a lyophilized cake or an aqueous solution. Further, in certainembodiments, the Nodal inhibitor product may be formulated as alyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions of the invention can be selected forparenteral delivery. Alternatively, the compositions may be selected forinhalation or for delivery through the digestive tract, such as orally.Preparation of such pharmaceutically acceptable compositions is withinthe skill of the art.

The formulation components are present preferably in concentrations thatare acceptable to the site of administration. In certain embodiments,buffers are used to maintain the composition at physiological pH or at aslightly lower pH, typically within a pH range of from about 5 to about8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be provided in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired Nodal inhibitor in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which the Nodal inhibitor is formulated as a sterile,isotonic solution, properly preserved. In certain embodiments, thepreparation can involve the formulation of the desired molecule with anagent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that may provide controlled or sustained release ofthe product which can be delivered via depot injection. In certainembodiments, hyaluronic acid may also be used to promote sustainedduration in the circulation. In certain embodiments, implantable drugdelivery devices may be used to introduce the desired Nodal inhibitor.

Pharmaceutical compositions of the invention can be formulated forinhalation. In these embodiments, Nodal inhibitors are advantageouslyformulated as a dry, inhalable powder. In preferred embodiments, Nodalinhibitor inhalation solutions may also be formulated with a propellantfor aerosol delivery. In certain embodiments, solutions may benebulized. Pulmonary administration and formulation methods thereforeare further described in International Patent Application No.PCT/US94/001875, which is incorporated by reference and describespulmonary delivery of chemically modified proteins.

It is also contemplated that formulations can be administered orally.Nodal inhibitors that are administered in this fashion can be formulatedwith or without carriers customarily used in the compounding of soliddosage forms such as tablets and capsules. In certain embodiments, acapsule may be designed to release the active portion of the formulationat the point in the gastrointestinal tract when bioavailability ismaximized and pre-systemic degradation is minimized. Additional agentscan be included to facilitate absorption of the Nodal inhibitor.Diluents, flavorings, low melting point waxes, vegetable oils,lubricants, suspending agents, tablet disintegrating agents, and bindersmay also be employed.

A pharmaceutical composition of the invention is preferably provided tocomprise an effective quantity of one or a plurality of Nodal inhibitorsin a mixture with non-toxic excipients that are suitable for themanufacture of tablets. By dissolving the tablets in sterile water, oranother appropriate vehicle, solutions may be prepared in unit-doseform. Suitable excipients include, but are not limited to, inertdiluents, such as calcium carbonate, sodium carbonate or bicarbonate,lactose, or calcium phosphate; or binding agents, such as starch,gelatin, or acacia; or lubricating agents such as magnesium stearate,stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving Nodal inhibitors insustained- or controlled-delivery formulations. Techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art. See,for example, International Patent Application No. PCT/US93/00829, whichis incorporated by reference and describes controlled release of porouspolymeric microparticles for delivery of pharmaceutical compositions.Sustained-release preparations may include semipermeable polymermatrices in the form of shaped articles, e.g. films, or microcapsules.Sustained release matrices may include polyesters, hydrogels,polylactides (as disclosed in U.S. Pat. No. 3,773,919 and EuropeanPatent Application Publication No. EP 058481, each of which isincorporated by reference), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22:547-556),poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed.Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105),ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (European Patent Application PublicationNo. EP 133,988). Sustained release compositions may also includeliposomes that can be prepared by any of several methods known in theart. See e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA82:3688-3692; European Patent Application Publication Nos. EP 036,676;EP 088,046 and EP 143,949, incorporated by reference.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method may beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or as a dehydrated or lyophilized powder. Such formulations may bestored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration.

Nodal inhibitors useful in the methods of the invention can be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption ina patient, using methods that are well known in the pharmaceutical arts.

The Nodal inhibitors may be administered orally, topically,parenterally, by inhalation or spray or rectally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles. The term parenteral as usedherein includes percutaneous, subcutaneous, intravascular (e.g.,intravenous), intramuscular, or intrathecal injection or infusiontechniques and the like.

Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents and preservative agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain theactive ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients that are suitable for the manufacture of tablets.These excipients may be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example starch, gelatin oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques. In some cases such coatings may be prepared by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatincapsules, wherein the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, oras soft gelatin capsules wherein the active ingredient is mixed withwater or an oil medium, for example peanut oil, liquid paraffin or oliveoil.

Formulations for oral use may also be presented as lozenges.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents may beadded to provide palatable oral preparations. These compositions may bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Pharmaceutical compositions of the invention may also be in the form ofoil-in-water emulsions. The oily phase may be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations may also contain a demulcent, a preservative, flavoringand/or coloring agents. The pharmaceutical compositions may be in theform of a sterile injectable aqueous or oleaginous suspension. Thissuspension may be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The Nodal inhibitors may also be administered in the form ofsuppositories, e.g., for rectal administration of the drug. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nodal inhibitors may be administered parenterally in a sterile medium.The drug, depending on the vehicle and concentration used, can either besuspended or dissolved in the vehicle. Advantageously, adjuvants such aslocal anesthetics, preservatives and buffering agents can be dissolvedin the vehicle.

The formulations can also be preferably applied as a topical gel, spray,ointment or cream, or as a suppository, containing the activeingredients in a total amount of, for example, 0.075 to 30% w/w,preferably 0.2 to 20% w/w and most preferably 0.4 to 15% w/w. Whenformulated in an ointment, the active ingredients may be employed witheither paraffinic or a water-miscible ointment base.

Alternatively, the active ingredients may be formulated in a cream withan oil-in-water cream base. If desired, the aqueous phase of the creambase may include, for example at least 30% w/w of a polyhydric alcoholsuch as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol,polyethylene glycol and mixtures thereof. The topical formulation maydesirably include a compound which enhances absorption or penetration ofthe active ingredient through the skin or other affected areas. Examplesof such dermal penetration enhancers include dimethylsulfoxide andrelated analogs.

The compounds of this invention can also be administered by atransdermal device. Preferably topical administration will beaccomplished using a patch either of the reservoir and porous membranetype or of a solid matrix variety. In either case, the active agent isdelivered continuously from the reservoir or microcapsules through amembrane into the active agent permeable adhesive, which is in contactwith the skin or mucosa of the recipient. If the active agent isabsorbed through the skin, a controlled and predetermined flow of theactive agent is administered to the recipient. In the case ofmicrocapsules, the encapsulating agent may also function as themembrane. The transdermal patch may include the compound in a suitablesolvent system with an adhesive system, such as an acrylic emulsion, anda polyester patch.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner. While the phase may comprisemerely an emulsifier, it may comprise a mixture of at least oneemulsifier with a fat or an oil or with both a fat and an oil.Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. It is also preferredto include both an oil and a fat. Together, the emulsifier(s) with orwithout stabilizer(s) make-up the so-called emulsifying wax, and the waxtogether with the oil and fat make up the so-called emulsifying ointmentbase which forms the oily dispersed phase of the cream formulations.Emulsifiers and emulsion stabilizers suitable for use in the formulationof the present invention include Tween 60, Span 80, cetostearyl alcohol,myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate,among others.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties, since the solubility of theactive compound in most oils likely to be used in pharmaceuticalemulsion formulations is very low. Thus, the cream should preferably bea non-greasy, non-staining and washable product with suitableconsistency to avoid leakage from tubes or other containers. Straight orbranched chain, mono- or dibasic alkyl esters such as di-isoadipate,isocetyl stearate, propylene glycol diester of coconut fatty acids,isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters may be used.These may be used alone or in combination depending on the propertiesrequired. Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils can be used.

For therapeutic purposes, the Nodal inhibitors of this invention areordinarily combined with one or more adjuvants appropriate to theindicated route of administration. If administered by mouth, thecompounds may be admixed with lactose, sucrose, starch powder, celluloseesters of alkanoic acids, cellulose alkyl esters, talc, stearic acid,magnesium stearate, magnesium oxide, sodium and calcium salts ofphosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate,polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted orencapsulated for convenient administration. Such capsules or tablets maycontain a controlled-release formulation as may be provided in adispersion of active compound in hydroxypropylmethyl cellulose.Formulations for parenteral administration may be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions and suspensions may be prepared from sterile powders orgranules having one or more of the carriers or diluents mentioned foruse in the formulations for oral administration. The compounds may bedissolved in water, polyethylene glycol, propylene glycol, ethanol, cornoil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodiumchloride, and/or various buffers. Other adjuvants and modes ofadministration are well and widely known in the pharmaceutical art.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 14 g per patient perday). The amount of active ingredient that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration. Dosageunit forms will generally contain between from about 1 mg to about 500mg of an active ingredient. The daily dose can be administered in one tofour doses per day. In the case of skin conditions, it may be preferableto apply a topical preparation of compounds of this invention to theaffected area two to four times a day.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination and the severityof the particular disease undergoing therapy.

For administration to non-human animals, the composition may also beadded to the animal feed or drinking water. It may be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It may also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

Dosing frequency will depend upon the pharmacokinetic parameters of theparticular Nodal inhibitor used in the formulation. Typically, aclinician administers the composition until a dosage is reached thatachieves the desired effect. The composition may therefore beadministered as a single dose, or as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages may be ascertained through use ofappropriate dose-response data. In certain embodiments, Nodal inhibitorscan be administered to patients throughout an extended time period.

Pharmaceutical compositions and/or Nodal inhibitors can be administeredalone or in combination with other therapeutic agents, in particular, incombination with other chemotherapeutic agents.

In addition, the invention provides methods for monitoring theeffectiveness of a pharmaceutical composition as an agent for treatingaggressive melanoma in a patient comprising the steps of: (a) obtaininga sample of skin cells from a patient; (b) assaying the skin cells forthe presence of Nodal; (c) administering an amount a pharmaceuticalcomposition to the patient; (d) repeating step (a) using asubsequently-collected biological sample obtained from the patient; and(e) comparing the amount of Nodal detected in the skin cells from step(a) with the amount of Nodal detected in the skin cells from step (c),wherein the effectiveness of the pharmaceutical composition is monitoredby detecting changes in the amount of Nodal in thesubsequently-collected skin cells compared with the skin cells taken instep (a).

Unless otherwise required by context, singular terms used herein shallinclude pluralities and plural terms shall include the singular.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting the invention.

Example 1 3-D Matrices Preconditioned by Human Embryonic Stem CellsPromote Epigenetic Changes in Aggressive Tumor Cells

As illustrated in FIG. 1, H1 or HSF-6 human embryonic stem cells(hESCs), 5×10⁴ cells in compact colonies, were seeded onto a 3-D matrixcomprised of growth factor-reduced Matrigel (BD Biosciences) in thepresence of conditioned stem cell medium for 3 to 4 days. Subsequently,the hESCs were removed from their 3-D matrix with NH₄OH followed bythorough washes with double-distilled H₂O, PBS, and complete medium,leaving a denuded, preconditioned, 3-D matrix (CMTX Matrigel). Onto thispreconditioned matrix were seeded human amelanotic metastatic cutaneousmelanoma cells (C8161), 2.5×10⁵ cells/6-well dish, for 3 to 4 days. Atthe end of this incubation period, analyses of potential changes inmorphology, gene and protein expression, and behavioral function(s) wereperformed on the melanoma cells exposed to the hESC preconditionedmatrix microenvironment. Preconditioning of an extracellular matrixexerted a dramatic effect on melanoma cell morphology (shown in FIG. 2).On a control unconditioned Matrigel matrix, C8161 melanoma cells (FIG.2A) grew into overconfluent monolayers, whereas undifferentiated H1(FIG. 2B) and HSF-6 (FIG. 2C) human embryonic stem cells (hESCs) formedcompact colonies of cells with a high nucleus-to-cytoplasm ratio.However, C8161 melanoma cells seeded onto the 3-D matricespreconditioned by the human embryonic stem cells acquired an alteredphenotype manifested by the formation of spheroids similar to thecolonies formed by human embryonic stem cells (FIG. 2D-F). In contrast,the conditioned media from human embryonic stem cells did not exert anepigenetic change on the C8161 cells (FIG. 2G), suggesting that hESCsinfluence melanoma cell phenotype through the alteration of theimmediate microenvironment.

To further analyze epigenetic changes in the phenotype of C8161 cellsexposed to the human embryonic stem cell microenvironment, Western blotand RT-PCR analyses of a melanocyte marker, Melan-A were performed(FIGS. 3A and 3B). Melan-A was absent both in H1 human embryonic stemcells (indicating their lack of a differentiated pigment cell phenotype)and in C8161 melanoma cells (illustrating their dedifferentiatedphenotype) on Matrigel. However, Melan-A expression was induced inamelanotic C8161 melanoma cells exposed to the H1 preconditionedMatrigel matrix (FIG. 3A), demonstrating the epigenetic induction of amelanocyte-specific phenotype marker, similar to control melanocytes(HEMn) on Matrigel. By contrast, C8161 melanoma cells exposed to 3-Dmatrices preconditioned by normal HEMn were not induced to change theirmorphology or to express Melan-A (FIG. 3B). Thus, the normal melanocytemicroenvironment does not share the ability of the hESC microenvironmentto epigenetically reprogram metastatic melanoma cells to express amelanocyte-like phenotype. This was confirmed by the results of FIG. 3C,showing semi-quantitative RT-PCR analysis which demonstrates that acollagen I 3-D matrix preconditioned by human melanocytes (HEMn18 orHEMn20 CMTX) does not change the expression of the genes tested,indicating that the benign melanocyte microenvironment does notepigenetically influence metastatic melanoma cells to change theirplastic, molecular phenotype.

Example 2 Aggressive Tumor Cells are Less Invasive and TumorigenicFollowing Culture on hESC Microenvironments

The aggressiveness of tumor cells is correlated with their ability toinvade through the extracellular matrix; thus, the effect of hESCmicroenvironments on melanoma cell invasion was investigated. Asillustrated in FIG. 4A, the in vitro invasiveness of aggressive C8161cells was significantly inhibited following culture on matricespreconditioned by hESCs, suggesting suppressive, anti-invasion cuesassociated with this human embryonic microenvironment.

Comparable results were found in vivo tumor formation. Amicroenvironment of human embryonic stem cells (H9CMTX) was prepared asdescribed above. C8161 human cutaneous melanoma cells were exposed tothe H9CMTX or Matrigel for 3 to 4 days prior to transplantation in amouse model. Nude immunocompromised mice received an injection of theC8161 cells subcutaneously into the midscapular region (to mimicspontaneous metastatic dissemination found in human cancers). Theanimals were injected using a 25 or 27-gauge needle with 2.5×10⁵ tumorcells/mouse in 0.05 ml RPMI media.

Tumor size was monitored on alternate days and was measured using amicrocaliper. At the time of necropsy (19 days after injection), themice were euthanized using CO₂ compressed gas asphyxiation followed bycervical dislocation and the tumor and major organs were removed andprepared for histology. The sections were stained with anti-Nodalantibodies (R&D Systems) to determine Nodal expression in the tumors(see Example 6). As shown in FIGS. 4A and 4B, melanoma cells were lessinvasive and tumorigenic in vivo following culture on hESCmicroenvironments.

Example 3 Characterization of Nodal Signaling Pathway Members in HumanNormal Cells, Metastatic Cancer Cells, Human Embryonic Stem Cells andOther Stem Cell Types: Aggressive Tumors Express Nodal but not Lefty

In order to elucidate the expression of key components of the Nodalsignaling pathway in normal, neoplastic and stem cell types, Westernblot analyses were conducted which revealed that in a manner similar tohESCs, metastatic melanoma (C8161) and breast carcinoma (MDA-MB-231)cells express Nodal protein at approximately 48 kDA (FIG. 5A). This isin contrast to corresponding normal cell types [melanocytes, myoepethialcells (Hs 578 Bst) and primary human mammary epithelial cells (HMEpC)],in which Nodal was not detected.

The Lefty proteins (Lefty-A, Lefty-B), divergent members of the TGF-βsuperfamily, spatially and temporally antagonize Nodal in embryologicalsystems (Tabibzadeh et al., 2006, Stem Cells 24:1998-2006). Moreover,the Lefty genes are downstream targets of Nodal signaling, therebyproviding a powerful negative-feedback loop for this pathway. Id. UsingWestern blot analysis it was determined that hESCs express Lefty proteinat approximately 42, 34 and 28 kDAs. In contrast, Lefty is not expressedby metastatic breast carcinoma and melanoma cells or by correspondingnormal somatic cell types (FIG. 5A). Real time RT-PCR analysis confirmedthese results as Lefty mRNA expression was exclusive to the hESC celllines (FIG. 5B).

Nodal propagates its signal by binding to heterodimeric complexesbetween type I (ALK 4/7) and type II (ActRIIB) activin-like kinasereceptors. Genetic studies in zebrafish and mice have determined thatCripto, an Epidermal Growth Factor-Cripto-1/FRL1/cryptic (EGF-CFC)family member, directly associates with ALK 4 and Nodal and that theseassociations facilitate the ability of Nodal to propagate its signal(Schier et al.; Yeo et. al., 2001, Mol. Cell. 7:949-957). Using Westernblot analysis and immunofluorescence microscopy, it was determined thathESCs uniformly express high levels of Cripto at approximately 35 kDA;however, only a subpopulation of metastatic human melanoma (C8161) andbreast carcinoma (MDA-MB-231) cells express a relatively low level ofCripto (FIG. 5A,C).

In order to analyze the expression of Nodal, Lefty and Cripto in otherhuman stem cell types and in first trimester human cytotrophoblast cells(HTR-8/SVneo), Western blot analyses were conducted which revealed thatumbilical cord derived mesenchymal stem cells (MSC; SC00125) and adultMSCs do not express Nodal and Cripto, and that although amnioticfluid-derived stem cells (GM00473, GM00957A) and cytotrophoblast cellsexpress Cripto, only the latter developmental cell type expresses anappreciable amount of Nodal (FIG. 5D). Of note, in contrast to hESCs,none of the other stem cell lines examined expressed an appreciablelevel of Lefty protein or mRNA (FIG. 5B).

In summary, like hESCs, cancer cells express Nodal, while unlike hESCs,they do not express Lefty. C8161 cells (human metastatic melanoma cells)and MDA-MB-231 cells (human metastatic breast carcinoma cells) expressedNodal and Cripto (at a low level), and they did not express Lefty.Expression of Nodal, Lefty, and Cripto was undetectable in normal humanmelanocytes, Hs 578 Bst normal human myoepithelial cells, and HMEpCnormal human mammary epithelial cells.

Example 4 Nodal Expression Correlates with Tumor Progression

Human melanoma specimens were screened for the presence of Nodalprotein. Formalin-fixed, paraffin-embedded archival tissue was obtainedfrom patients with primary or metastatic cutaneous melanoma (LoyolaUniversity Chicago, Ill.). Immunohistochemical staining was performed ona FINS 710i Automated Immunostainer (Richard-Allan Scientific (RAS),Kalamazoo, Mich.) with the Multi-Species HRP/AEC Detection Systems.Following deparaffinization in xylene, ethanol degradation, and antigenretrieval with citrate buffer, four blocking steps were applied: 0.03%hydrogen peroxide, Avidin and Biotin blocks (Avidin/Biotin blocking kit,Vector Laboratories, Inc., Burlingame, Calif.), and a Serum-Free proteinblock. Anti-Nodal antibody (20 μg/mL, R&D) was applied for 90 minutes.Slides were rinsed in TBS-T, incubated with biotinylated anti-goat IgG(2 μg/ml, Vector Labs), washed with TBS-T and incubated with thestreptavidin peroxidase reagent for 15 minutes. Color was produced withAEC (red) substrate (RAS) and counterstaining with Mayer's hematoxylin.Samples were dehydrated in reagent grade alcohol and cover slipped withpermanent mounting medium. Negative control reactions were conductedwith ChromPure Goat IgG (Jackson Labs), isotype matched and used at thesame concentration as the Nodal antibody.

The immunohistochemistry demonstrated that Nodal is absent in normalskin (FIG. 6A) and is weakly expressed or absent in primary melanomas(FIG. 6B). In the primary lesions, Nodal immunostaining was generallyconfined to small clusters of tumor cells in the vertical growth phaseand was rarely observed in radial lesions (n=5; FIGS. 6C and D). Incontrast, Nodal protein was expressed in 60% of the cutaneous melanomametastases examined (n=10; FIGS. 6E and F). Immunostaining washeterogeneous, varying among patients in extent, intensity andlocalization. For example, Nodal was found localized to cell membranes,and was expressed diffusely in the cytoplasm (FIG. 10). Western blotanalyses similarly revealed that 45% of metastatic melanomas tested werepositive for Nodal (n=22). This is in contrast to normal skin (n=9), andmelanocytes (n=5), neither of which expressed Nodal (data summarized inTable 1). Collectively, these results demonstrated that Nodal expressionwas positively correlated with melanoma progression.

TABLE 1 Nodal Nodal Staining Signal (IHC) Case Type (Western) NormalSkin − Normal Skin − Normal Skin − Normal Skin − Normal Skin − NormalSkin − Normal Skin − Normal Skin − Normal Skin − Melanocytes −Melanocytes − Melanocytes − Primary − Melanocytes − Primary −Melanocytes − Primary  −* Dendritic Cells − Primary  −* Dendritic Cells− Primary  −* Metastatic Melanoma − Metastasis  −* Metastatic Melanoma −Metastasis ++ Metastatic Melanoma + Metastasis + Metastatic Melanoma +Metastasis + Metastatic Melanoma − Metastasis − Metastatic Melanoma −Metastasis − Metastatic Melanoma − Metastasis − Metastatic Melanoma +Metastasis ++ Metastatic Melanoma − Metastasis ++ Metastatic Melanoma −Metastasis ++ Metastatic Melanoma + Metastatic Melanoma − MetastaticMelanoma + Metastatic Melanoma − Metastatic Melanoma − MetastaticMelanoma + Metastatic Melanoma − Metastatic Melanoma + MetastaticMelanoma + Metastatic Melanoma + Metastatic Melanoma − MetastaticMelanoma + ++ Represents strong positive staining for Nodalencompassing >75% of the tumor mass + Represents positive staining forNodal encompassing >50% of the tumor mass or the detection of Nodalusing Western Blot analyses −* Represents Nodal staining in a smallsubpopulation (<10%) of the tumor mass − Represents the absence of Nodal

As with the positive correlation of Nodal expression with melanomaprogression, such that Nodal protein is not expressed in normalmelanocytes or radial growth phase melanomas, but is present in moreaggressive vertical growth phase and metastatic lesions,immunohistochemical analysis of a human breast tissue microarray (TMA)revealed that Nodal protein is similarly absent in normal breast tissue,and that its expression is positively correlated with breast carcinomaprogression (FIG. 7).

The expression and prevalence of Nodal staining in breast tissue wasdesignated as none, weak (<25%), moderate (25-75%) or strong (>75%).DCIS is ductal carcinoma in situ and IDC is invasive ductal carcinoma.Spearman's rank correlation showed a significant positive correlationbetween breast cancer progression and Nodal expression (P<0.05) (datasummarized in Table 2).

TABLE 2 None Weak Moderate Strong Total Benign 26 1 0 0 27 DCIS 3 0 0 03 IDC 24 1 3 5 33

Example 5 Localization of Nodal and Lefty in hESC Matrices

Immunofluorescence localization with confocal microscopy was performedin order to visualize the deposition of Lefty into the microenvironmentof hESCs. Utilizing this methodology, it was determined that Leftyprotein localizes to the areas where hESCs are in contact with theunderlying Matrigel matrix, and that hESC-derived Lefty permeates intothe underlying matrix (FIG. 8). This is in contrast to Nodal protein,which localizes to the surface of hESC colonies. These results wereconfirmed with Western blot analyses, which demonstrated that Leftyprotein can be detected in Matrigel conditioned by H9 hESCs (H9CMTX;FIG. 8), but that Nodal protein is not detectable in this H9CMTX.Furthermore, neither Nodal nor Lefty were found in unconditioned controlMatrigel alone.

Example 6 Nodal Expression Down-Regulated in Aggressive Tumor CellsExposed to hESC Conditioned Matrix

A determination of the effects of H9CMTX on Nodal expression inmetastatic melanoma (C8161) and breast carcinoma (MDA-MB-231) cells wasundertaken. As illustrated in FIG. 9, the microenvironment of humanembryonic stem cells (hESCs) leads to the reduction of Nodal expressionand tumorgenicity in plastic metastatic melanoma and breast cancer cellsexposed to the embryonic preconditioned matrix. Western blot analyses(see below) revealed that the microenvironments of hESCs reduced theexpression of Nodal protein in multipotent melanoma (C8161) and breastcarcinoma (MDA-MB-231) cells (FIG. 9A). Exposure to H9CMTXdown-regulates Nodal protein expression in both melanoma and breastcarcinoma cells, and this effect is reversible over time (FIG. 9B).Exposure to H9CMTX similarly abrogates Nodal mRNA expression in themelanoma and breast carcinoma cells (FIG. 9C).

FIG. 9D shows immunohistochemical analysis of Nodal staining in anorthotopic tumor derived from C8161 cells pre-exposed to Matrigel or ahESC conditioned matrix (H9CMTX) (from in vivo experiment in Example 2).The C8161 cells exposed to the H9CMTX expressed much less Nodal.

As a functional correlate, it was determined that exposure of C8161 andMDA-MB-231 cells to H9CTMX results in a significant reduction in theirability to undergo anchorage independent growth, and that thisinhibition of in vitro clonogenicity can be partially rescued by theinclusion of recombinant Nodal (100 ng/mL) (FIG. 9E). Of note, usingWestern blot analysis in conjunction with real time RT-PCR, the abilityto inhibit Nodal expression in cancer cells was shown to be exclusive tothe microenvironment of hESCs (FIGS. 9F & 9G). For example, exposure ofC8161 cells to matrices conditioned by melanocytes, amniotic fluidderived stem cells (GM00473, GM00957A), or cytotrophoblast cells(HTR-8/SVneo) did not inhibit Nodal protein or mRNA expression (FIGS. 9F& 9G), thus illuminating the exclusivity of the epigenetic influence ofthe hESC microenvironment.

Western Blot Analyses

Protein lysates were prepared and quantified as previously described inHess et al., 2001, Cancer Res. 61:3250-3255. Equal amounts of proteinwere separated by SDS-polyacrylamide gel electrophoresis under reducingconditions, and the resolved proteins were transferred onto Immobilon-Pmembranes (Millipore Corp., Bedford, Mass.). Membranes were blocked in1% TBS, 0.1% Tween 20 (TBS-T) and 5% dry milk powder or 3% gelatin (forNodal Westerns). Blots were incubated with anti-Nodal or anti-Leftyantibodies (Polyclonal rabbit anti-Nodal (H-110) 1:500 Santa CruzBiotechnology, Santa Cruz, Calif.; Polyclonal goat anti-Lefty 1:500 R&DSystems, Minneapolis, Minn.), washed in TBS-T, and incubated with theappropriate horseradish peroxidase-labeled secondary antibody. Secondaryantibodies were detected by enhanced chemiluminescence (Super Signal;Pierce, Rockford, Ill.) and exposure to autoradiography film (MolecularTechnologies, St Louis, Mo.). Nodal protein was detected as two majorbands at ˜48 and 35 kDa representing precursor and pro-Nodalrespectively. Nodal often appeared as multiple bands, likely due todegradation of protein modifications. All experiments were done at leastthree times.

Example 7 Lefty is a Major hESC-derived Factor Responsible forInhibiting Nodal Expression and Clonogenicity in Metastatic Cancer Cells

As noted previously, the microenvironment of human embryonic stem cells(hESCs) leads to the reduction of Nodal expression and tumorgenicity inplastic metastatic melanoma and breast cancer cells exposed to theembryonic preconditioned matrix. It was determined that there is anabundance of the Nodal inhibitor Lefty within the hESC conditionedmatrices (FIG. 10A; CMTX). By contrast, Lefty protein was absent in theC8161 cells on its own conditioned matrix (FIG. 10A; C8161+CMTX).

Cancer cells were also exposed to Matrigel conditioned by hESCs in whichLefty protein expression was knocked down with FITC-tagged Morpholinooligonucleotides specific for Lefty-A and Lefty-B (MO^(LEFTY)). Thefluorescently-tagged Morpholinos could be detected microscopically inover 75% of the hESC colonies treated (FIG. 10B), and Western blotanalysis confirmed the efficient knock down of Lefty protein in hESCsfor up to 3 days (FIG. 10C). The expression of Oct-3/4 and Nanog,representative of hESC pluripotency, was not affected during this time,and the morphology of the hESC colonies was not altered. Thus, althoughMO^(LEFTY) efficiently knocked down Lefty protein expression in thehESCs, it did not induce stem cell differentiation (FIG. 10D). Real timeRT-PCR analysis revealed that exposure of metastatic melanoma cells toMatrigel conditioned by H9 hESCs treated with MO^(LEFTY) did not resultin an abrogation of Nodal expression (FIG. 10E). In fact this “H9Lefty-deficient” matrix up-regulated Nodal mRNA expression in the C8161cells (FIG. 10E).

Additionally, Dynabeads covalently coupled to anti-Lefty antibody wereutilized to isolate Lefty from hESCs cultured on a feeder-free Matrigelmatrix. This purified hESC-derived Lefty was subsequently seeded intofresh Matrigel and the effects of the “Lefty-containing” matrix oncancer cell phenotype were examined. Western blot analysis revealed thathESC-derived Lefty abrogates and diminishes Nodal protein expression inmetastatic melanoma (C8161) and breast carcinoma (MDA-MB-231) cells,respectively (FIG. 10F).

Also, exposure of C8161 and MDA-MB-231 cells to “H9 Lefty-containing”matrix was found to significantly reduce anchorage independent growth,and this inhibition of in vitro clonogenicity could be completelyrescued by the inclusion of recombinant Nodal (100 ng/mL) (FIG. 10G).

Example 8 rLefty Capable of Inhibiting Nodal at Elevated Concentrations

As shown in the Western blot in FIG. 11, addition of rLefty to C8161cells reduces Nodal expression at the concentrations shown. rLeftyB caninhibit Nodal protein in C8161 cells, but at a higher doses than itshESC counterpart. These results are consistent with prior findings(Tabibzadeh, S et al., 2006, Stem Cells 24: 1998-2006).

Example 9 Lefty Derived from hESC's, Unlike Recombinant Lefty, isGlycosylated

In an effort to understand the disparate results between hESC-derivedLefty and rLefty on Nodal signaling, an analysis of glycoprotein contentin rLefty-B, rLefty-A and a lysate from the H9 hESCs plus theirconditioned matrix was undertaking. It was found that in contrast to therLefty proteins, H9-derived Lefty is heavily glycosylated (FIG. 12).

Example 10 Nodal Inhibition and the Microenvironment of hESCs AbrogateTumorigenicity In Vivo

The effects of the hESC microenvironment on the in vivo tumorigenicityof melanoma and breast carcinoma cells were examined using orthotopicmouse models. Exposure of metastatic melanoma (C8161) and breastcarcinoma (MDA-MB-231) cells to H9CMTX resulted in a significantreduction in tumorigenicity as compared to cells exposed tounconditioned Matrigel (FIGS. 4B & 13A). In order to furthersubstantiate the role of Nodal in tumorigenicity, and to illuminate apotential mechanism for the tumor-suppressive properties of the hESCmicroenvironment, previously shown to diminish Nodal expression (FIG.9), orthotopic mouse models were utilized to examine the effects of: (a)Ectopic Nodal expression on the tumorigenic potential of C81-61 cells(isogenetically matched non-tumorigenic variants of the metastatic C8161melanoma cell line); and (b) Nodal inhibition on MDA-MB-231 breastcarcinoma tumorigenicity. The control C81-61 melanoma cell line wasunable to form palpable tumors when 500,000 cells were injected, andusing gross observation and histology 5.5 weeks after inoculation, thesetumor cells could not be detected at the site of injection. In contrast,100% of C81-61 cells transfected with a Nodal expression vector formedpalpable tumors within 3.5 weeks of injection (FIG. 13B). Palpabletumors arose within 2.5 weeks after the injection of 500,000 controlMDA-MB-231 cells, and knocking down Nodal expression with NodalMorpholinos (MO^(Nodal)) resulted in a significant reduction inMDA-MB-231 tumorigenicity (FIG. 13C) when the same number of cells wereinjected.

In order to establish a mechanism by which exposure to the hESCmicroenvironment abrogates tumorigenicity, the effects of this treatmenton the in vivo tumor cell proliferation-to-apoptosis ratio wereanalyzed. Using immunohistochemical staining for Ki67 as a measure ofproliferation, and terminal deoxynucleotidyl transferase biotin-dUTPnick-end labeling (TUNEL) as a measure of apoptosis, it was determinedthat Nodal knock down and exposure to hESC CMTX correspondinglydecreased the ratio of proliferation to apoptosis in metastatic C8161melanoma cells and in metastatic MDA-MB-231 breast carcinoma cells(FIGS. 13DE & 13E). Moreover, an in vitro analysis of cell proliferationdemonstrated reduced proliferation in C8161 and MDA-MB-231 cells treatedwith MO^(Nodal) relative to cells treated with MO^(Control) (FIG. 13F).

Example 11 Nodal Signaling Essential for Tumor Formation; Inhibitors ofNodal Signaling Reduce Aggressiveness and Tumorigenicty

The role of Nodal signaling in tumor formation was analyzed, and it wasfound that downregulation of Nodal signaling results in acquisition of amelanocyte-like phenotype and loss of the dedifferentiated, plasticphenotype.

Administration of an anti-Nodal Morpholino (MO Nodal) also resulted indown-regulation of Nodal, and an in vivo reduction in tumor formation.An in vitro colony forming assay was used to analyze colony-formingability of poorly aggressive C81-61 cells, aggressive C8161 cells, C8161cells treated with MO_(Nodal) and C8161 cells treated with MO_(Nodal)and rescued with recombinant Nodal (100 ng/mL). The assay was conductedusing 50,000 cells suspended in 0.35% agarose in RPMI containing 10%serum, which were plated into 6-well dishes on 0.5% agar in the samemedium. Colonies grew, and pictures were taken at day 7. After 2 weeks,colonies were stained with Crystal Violet and counted.

Utilizing the in vitro assay, it was found that C8161 cells were able toform colonies in soft agar within 7 days, and that their less aggressiveisogenic counterparts (C81-61) were not clonogenic (FIG. 14A). Nodalinhibition with MO_(Nodal) significantly diminished the ability of C8161cells to undergo anchorage-independent growth. Even after 2 weeks,MO_(Nodal) reduced the colony formation of the C8161 cells by 57% (n=16,p<0.001), a phenomenon that was rescued by the inclusion of rNodal (100ng/mL). Interestingly, rNodal did not induce colony formation in theC81-61 cell line. For the clonogenic assays, statistical significancewas determined using a test. In all cases, differences werestatistically significant at p<0.05.

As a corollary to these findings, an orthotopic mouse model was used toexamine the effect of Nodal inhibition on melanoma tumor formation. Forthe experimental tumourigenesis model, 5 week old nude mice (Harlan,Madison, Wis.) were injected subcutaneously with 250,000 C8161 cells,treated with control or anti-Nodal Morpholinos, in 50 μL of completeRPMI. Tumor measurements were taken on days 3, 7, 14 and 17post-injection, and mice were sacrificed on day 17. In vivo tumorformation in a mouse injected with C8161 cells treated with eitherMO_(Control) or MO_(Nodal) is shown in FIG. 14B. For the orthotopicmouse tumor formation studies, statistical significance was determinedusing the Kruskal-Wallis One Way Analysis of Variance on Ranks, followedby Dunn's method.

Palpable subcutaneous tumors arose within 7 days following the injectionof only 250,000 control C8161 cells. In contrast, knocking down Nodalexpression resulted in a significant reduction in C8161 tumorigenicity(FIG. 12B; n=5, p<0.05) when the same number of cells were injected.This reduction in tumorgenicity was characterized by 30% diminution oftumor incidence as well as a decrease in tumor growth.

Immunohistochemistry was used to analyze tumors from the mice, whichshowed that the tumors that formed in the MO_(Nodal) treatment groupstarted to regain Nodal expression by day 17 (FIG. 14C).

Additionally, poorly aggressive melanoma cells (C81-61) cells acquiredtumorigenic potential when transfected with Nodal cDNA. C81-61 cellswere transfected with either an empty vector or a Nodal expressionconstruct (n=5). As shown in FIG. 14D, such cells demonstrated tumorgrowth.

Administration of an ALK 4/5/7 inhibitor resulted in a reduction of theexpression of various vasculogenic mimicry plasticity biomarkers. FIG.15A shows Western blot analyses of Nodal, phosphorylated SMAD-2, totalSMAD 2/3, and Actin in C8161 cells 48 hours after administration ofeither vehicle or an ALK 4/5/7 inhibitor (SB431542, 1 μM, 10 μM). AllNodal bands represent the pro-protein. FIG. 15B shows Western blotanalyses of Nodal, Tyrosinase and Actin in C8161 cells 24 hours afterthe administration of either vehicle or different concentrations of ALKinhibitor while FIG. 15C shows a Western analyses for VE-Cadherin,Keratin 18 and Actin in C8161 cells cultured on 3-D collagen I matricesfor 6 days in the presence of vehicle or different concentrations of ALKinhibitor. FIG. 15D shows a reduction in invasive ability and FIG. 15Eshows an abrogation of vasculogenic mimicry following down-regulation ofNodal with ALK 4/5/7 inhibitor.

The experiments described above revealed that metastatic melanomasexpress the embryonic morphogen Nodal, that Nodal is essential for tumorformation, and that its effects can be mitigated through Nodal pathwayinhibition, either directly or indirectly (e.g. through ALK inhibition).

Example 12 Nodal Expression is Down-Regulated by Notch Inhibition

To address the possible molecular mechanisms underlying thereprogramming of melanoma cells exposed to the hESC matrixmicroenvironments, an analysis of the Nodal promoter was initiated, aputative binding sequence effector for the Notch pathway (CBF-1) wasdiscovered, and the possibility of molecular cross-talk between theNotch and Nodal pathways was investigated. Nodal expression inmetastatic melanoma cells treated with Notch siRNAs was knocked down,particularly with Notch 4 siRNA. Conversely, Notch expression wasrelatively unaffected by knockdown of Nodal, suggesting that Notch isupstream of Nodal with possible molecular cross-talk.

As shown in FIG. 16A, Nodal expression was knocked down in C8161 cells72 hrs following the administration of Notch siRNA. Real-time RT-PCR andWestern blot analyses confirmed the silencing of each Notch at this timepoint. In contrast, as shown in FIG. 16B, Notch expression is relativelyunaffected by the knockdown of Nodal (via treatment with the Nodalinhibitor SB431542) in C8161 cells.

Example 13 Hypermethylation Plays a Role in the Nodal Pathway inAggressive Tumor Cells

Hypermethylation of Nodal was observed in the highly metastatic C8161cells, but not in the isogenically matched C81-61 melanoma cells, nor inmelanocytes or hESCs (H9). Sequencing based methylation analyses,therefore, could be used to indicate the methylation status in humantumors, and hence serve as a valuable prognostic marker for diseasestate.

Nodal's methylation status is supported by work in Feinberg's laboratoryshowing that CTCF binding site methylation separates enhancers frompromoters. (Gius, et. al., 2004, Cancer Cell 6:361-371) In that work, itwas found that azacytidine shut down expression of as many genes as itactivated; it is known now that this subset of genes contains CTCFbinding sites within the promoter CpG island. In particular for Nodal,the sequence is CCGCGCTGGGTGCCCAG [SEQ ID NO: 1]. The consensus that wasidentified in genes activated by methylation isCCGCGN(N)GG(G)(N)GCC(N)CAG [SEQ ID NO:2], and Feinberg has directlydemonstrated methylation dependent activation, with CTCF insulatorbinding abrogation in several promoters with this consensus sequence.Paradoxically, when this site is methylated, CTCF can no longer bind,and the promoter is enabled. This is a major imprinting mechanism, andhas significant implications for how Nodal may be regulated during bothcancer and development.

Referring to FIG. 17, although culture of the C8161 cells in thepresence of a hESC microenvironment (H9CMTX) globally increasesmethylation by only 6.8%, the shaded region has a 32% increase inmethylation when cells are cultured on H9CMTX versus Matrigel alone.Sequence alignment revealed that the differentially methylated cytosinesare associated with putative transcription factor binding sites. Theshaded area contains consensus sequences for EBP, Sp 1 and AP-2alpha,and the 32% increase in methylation in this region may indicate asilencing of the Nodal gene in the tumor cells exposed to the hESC CMTX.

Example 14 Exemplary Assays and Procedures

General Maintenance of Cell Lines

The derivation and phenotypic characteristics of the human melanoma celllines have been previously described. Seftor, et. al., 2002, Clin.Experim. Metastas. 19:233-246; Seftor, et. al., 2005 Cancer Res.65:10164-10169. The melanoma cell lines are maintained in RPMI 1640medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS,Gemini Bioproducts) and 0.1% gentamycin sulfate with the exception ofC81-61 cells which are maintained in Ham's F10 medium supplemented with15% FBS, 1× Mito+ (BD Bioscience) and gentamycin sulfate. Normal humanmelanocytes are purchased (Cascade Biologics) or isolated from neonatalforeskins. Seftor, et. al., 2005. A single cell suspension is prepared,added to plastic flasks for the adherence of melanocytes and the cellspropagated in Medium 254 with Human Melanocyte Growth Supplement(Cascade Biologics) including, 100 units/ml penicillin, 100 μg/mlstreptomycin, and 250 ng/mL amphotericin B. The human embryonic stemcell lines are cultured as previously described. Thomson, et. al., 1998,Science 282:1145-1147. Briefly, cells are grown in 6-well platesprecoated with 0.1% porcine gelatin and containing 1.9×105 irradiatedmouse embryonic fibroblasts (strain CF-1; ATCC) per well. The cells aremaintained in medium containing DMEM/F12 (1:1), 20% knock-out serumreplacement, non-essential minimal amino acids, L-glutamine(Invitrogen), β-mercaptomethanol, and 4 ng/ml FGF-2 (R&D Systems), andare split with collagenase (1 mg/ml) before the colonies begin tooverlap. The cultures are determined to be free of mycoplasmacontamination using a PCR-based assay (Roche). The normal human neonatalepidermal melanocytes (HEMn-LP; Cascade Biologics, Portland Oreg.),myoepithelial cells (Hs 578 Bst; American Type Culture Collection(ATCC), Manassas, Va.) and primary mammary epithelial cells (HMEpC; CellApplications Inc., San Diego Calif.) were maintained as per distributorinstructions. Live umbilical cord blood stem cells (SC00125; New JerseyStem Cell Resource at Coriell Institute for Medical Research) amnioticfluid derived stem cells (GM00473, GM00957A) and adult bone marrowderived mesenchymal stem cells (Stem Cell Technologies, Vancouver BC,Canada) were maintained under the recommended conditions. TheHTR-8/SVneo is a well characterized immortalized human extravillouscytotrophoblast cell line, and was maintained as previously describedGraham et al., 1993, Exp. Cell Res. 206:204-211. Recombinant Nodal andLefty (R&D Systems) were diluted as per manufacturer suggestions. Theexpression vector for wild type Nodal was kindly provided by Dr. DanielConstam (Swiss Institute for Experimental Cancer Research (ISREC),Epalinges, Switzerland) and was transfected into C81-61 cells aspreviously described. Le Good et al., 2005, Curr. Biol. 15:31-36.

Preparation/Preconditioning of 3-D Human Matrices

25-30 μl of a defined human matrix (50 μg/ml human laminin; 50 μg/mlhuman collagen IV in a 3 mg/ml human collagen I base; Sigma) are eitherspread onto coverslips or directly placed into 12-well culture dishesand polymerized with an application of 100% ethanol at room temperature.After extensive washes with PBS, hESC's are seeded onto the 3-D matrixin complete stem cell medium. After 3-4 days images are captureddigitally using a Zeiss Televal inverted microscope and Hitachi HV-C20CCD camera. The cells are then removed with 20 mM NH₄OH followed bythorough washes with sterile water, PBS and then complete medium. Theconditioned matrix is then analyzed by 2-D LDS-PAGE and Western blotdirectly, or reseeded with melanoma cells and incubated for anadditional 3 days. The cells re then harvested for further biochemical,molecular and functional analyses.

3-D Conditioned Matrix Experiments

Conditioned matrices were prepared using hESCs, melanocytes,myoepithelial cells, amniotic fluid derived stem cells, or trophoblastcells on growth factor-reduced Matrigel (14 mg/mL; BD Biosciences) aspreviously described. Postovit et al., 2006, Stem Cells 24: 501-505. Inall cases, cells were 80-100% confluent during the conditioning of thematrix. Alternatively, hESC-derived Lefty protein was seeded intoMatrigel prior to polymerization. Human melanoma (C8161) or breastcarcinoma (MDA-MB-231) cells, 2.5×10⁵ cells/6-well dish, weresubsequently exposed to this preconditioned matrix for 3 to 4 days.

Invasion/Migration Assay

The Membrane Invasion Culture System (MICS) chamber is used to evaluatethe degree of tumor cell invasion through matrices in vitro (bothstimulated and unstimulated) as described previously. Hendrix, M. J. C.et. al., 1992, J. Natl. Cancer Inst., 84:165-174.

Determination of Cell Viability and Proliferation

Cell proliferation is assayed by immunohistochemical staining of BrdUincorporation into newly synthesized DNA of replicating cells at varioustime points (BrdU Labeling and Detection Kit III; Roche). Assessment ofproliferation index is monitored by Ki-67 expression.

Proliferation Assay

1.5×10⁴ cells were plated in individual wells of a 24-well dish understandard tissue culture conditions and cell counts were taken dailyfollowing harvesting.

Soft Agar Clonogenic Assay

Clonogenicity of cells are assessed as previously described. Hamburger,A. W., and Salmon, S. E., 1977, Science 197:461-463. Each parameter istested in triplicate for clone formation in soft agar. Briefly, 104cells are plated in 60 mm Petri dishes in complete medium placed overthe soft agar. On specific days after the cells are plated, phasecontrast images of the colonies are taken using a Zeiss Axiovert 25 withan Hitachi HCV-20 color camera.

Anchorage Independent Growth Assays

Anchorage independent growth assays were conducted as previouslydescribed. Topczewska et al., 2006, Nat. Med. 12: 925-932.

Differentiation Assays of Stem Cell Populations and Clonally-DerivedMelanoma Stem Cells

Stem cells from the various 3-D preconditioned matrices are harvestedand replated on an appropriate ECM in a specified differentiation media,as previously described. Hendrix et. al., 2003, Nature Rev. Cancer3:411-421; Hsu et. al., 2004, Methods Mol. Med. 107:13-28; Pittenger et.al., 1999, Science 284:143-147

Experimental Orthotopic Tumor Models

5 week old mice were injected subcutaneously with 250,000 C8161 or500,000 C81-61 human cutaneous melanoma cells in 50 μL of complete RPMI;or 500,000 MDA-MB-231 cells in 50 μL of complete RPMI were injected intothe mammary fat pad of 8 week old mice. When tumors became palpablemeasurements were taken twice per week.

2-D L(ithium)DS-PAGE and Western Blot

Analysis of extracellular matrix components before and afterconditioning by the different cells is performed using Invitrogen's 3-10pH IPG strips in the first dimension and 4-12% Bis/TrisLDS-polyacrylamide gradient gels in the second dimension using MES(proteins up to ˜100 kDa) or MOPS (proteins>100 kDa) reservoir buffers,as per the manufacturer's protocols. The gels are stained with Sypro Redthen electroblotted onto Immobilon P membranes (Millipore) for Westernanalysis using specific extracellular matrix antibodies (Chemicon; R&DSystems; Life Technologies).

Recovery of Lefty from Cell Conditioned Matrices

M-280 tosylactivated Dynabeads (Dynal Biotech) are covalently coupled toanti-Lefty antibody (M−20:sc7408; Santa Cruz Biotechnology, Inc.) at afinal concentration of 8 μg antibody/1×107 beads as per themanufacturer's protocol. The cell conditioned matrix is solubilized inRIPA buffer, sonicated, centrifuged and the supernatant mixed byrotation with the beads for 1 hour at 4° C. After washing twice withPBS, Lefty is recovered using either 50 mM glycine-HCl (pH 3.0), thennormalized to pH 7.4 with a 0.1 volume of 1 M Tris pH 8.5, or 0.2 M TrispH8.5 plus 0.5 M NaCl.

RNA Extraction and Reverse Transcriptase Polymerase Chain Reaction(RT-PCR)

Total RNA was isolated using TRIzol reagent (Invitrogen) and 1 μg wasreverse transcribed as previously described. Topczewska et al., 2006.Real-time PCR was performed as previously described (Id.) using TaqMan®gene expression human primer/probe sets for the following genes: VEGF(Hs00173626 ml), TSP-1 (Hs00170236_m1), Ki67 (Hs00606991 ml), LeftyI/B(Hs00764128 μl), Nodal (Hs00250630_s1). Target gene expression wasnormalized to the endogenous control gene GAPDH (GAPDH: 4333764F), RPLPO(RPLPO: 4333761F) and/or 18S rRNA (Hs99999901_s1). Data was analyzedusing Applied Biosystems Sequence Detection Software (Version 1.2.3).

FACs Analysis/Sorting

Fluorescence Activated Cell (FAC) analyses and sorting are conductedusing the BD FACsAria. Prior to FAC analysis, cells are incubated withantibodies as per manufacturer instructions and intracellular proteinsdetected in cells that have been previously permeabilized. The FACprotocols are optimized for both cell surface proteins (such as CD34)and intracellular proteins (such as keratins). Live cell sorts areconducted using a 100 μm nozzle and aseptic technique. Successful livesorts are performed against cell surface proteins, fluorescently labeledcells and cells containing fluorescent anti-sense Morpholinos.

Glycoprotein Determination

Protein lysates underwent SDS-polyacrylamide gel electrophoresis andtransfer and were stained for glycoproteins using the Pro-Q Emerald 300staining kit (Molecular Probes). After drying the blot, glycoproteinswere visualized using an ultraviolet tansilluminator and an image of thegreen fluorescing proteins captured using a color CCD camera (Toshiba)equipped with a deep yellow #15 filter. The blot was then rehydrated asper the manufacturer's instructions and Lefty protein was detected withimmunoblotting.

IVIS Imaging System 200 Series

Real-time biophotonic imaging of GFP-labeled tumor cells in the mousemodel(s) is performed using a Xenogen IVIS Imaging System 200 Seriesimager. This system contains a custom lens and improved resolution withsingle cell sensitivity for in vitro analyses. A laser scanner andassociated software provides an ability to perform 3-D surfacetopography for single-view diffuse tomographic reconstructions ofinternal sources in order to track tumor formation and metastaticpotential of GFP-labeled tumor cells. Quantitative in vivo assays areperformed using dual reporters to differentiate increases in cellproliferation from increases in specific gene expression.

Laser Capture Microdissection

The Veritas Laser Capture Microdissection (LCM) system (Arcturus)combines a three objective lens microscope (up to 40×) for visualizing asample mounted on a slide and selecting the areas of interest, a UVlaser for cutting around the perimeter of the areas of interest, and aIR laser that melts and thereby sticks the surface of a collector cap tothese areas, or individual cells for isolation. The Veritas LCM can beused for isolating live cells cultured on 3-D matrices which have beencast in the etched space of a specially made membrane containing slidecompatible with the Veritas system (PEN frame slides). Captured materialis subsequently lysed for RNA isolation (Picopure, Arcturus) anddownstream applications including Q-PCR and microarray gene expressionanalysis.

Confocal Immuno-Microscopy

Immuno-confocal microcopy is performed using a Zeiss LSM 510 METAConfocal Microscope. Prior to analysis, 3-D cultures or tissue sectionsare incubated with specific antibodies against target proteins as perprotocols previously established in the laboratory.

In Situ Hybridization

3-D cultures or tissue samples are placed on subbed microscope slides,and prepared as previously described. Kulesa, et. al., 2000, Develop.127(13):2843-2852.

Nodal and Lefty Knockdown

Nodal, and Lefty protein expression were inhibited using anti-senseMorpholino oligonucleotides (Gene Tools Inc., Philomath, Oreg.). TheMorpholino sequences were selected based on manufacturer'srecommendations (21-25 mer antisense). Fluoroscein (FITC)-conjugatedcontrol (5′-CCTCTTACCTCAGTTACAATTTATA-3′) [SEQ ID NO: 19], Nodal(5′-AAGCAGCACCTCCAGCCCTTATATC-3′) [SEQ ID NO: 20], Lefty-A(5′-GCCACATGGTGCTGCCCTGGG-3′) [SEQ ID NO: 21], and Lefty-B(5′CTGCATGGTGCTGCCCTGGAGGA-3′) [SEQ ID NO: 22]. Morpholinos (20 μM) weredelivered using the scrape method. Topczewska et al., 2006. Cancer cellswere sorted for FITC and were recovered for 1 day prior toexperimentation.

Knockdown of Gene Expression by siRNA

Cells are plated in 6-well tissue culture plates and allowed to grow to50% confluence in serum containing, antibiotic-free medium. The cellsare then transfected with 10 or 100 nM of a gene-specific siRNA or anon-specific siRNA control using oligoFECTAMINE according tomanufacturer's specifications (Invitrogen). The cells are then harvested3 days post transfection and assessed for gene expression by RT-PCR,Q-PCR and Western blot analysis, as well as functional assays.Quantitative PCR (Q-PCR): Total RNA is isolated from cells using TrizolRNA isolation reagent (Invitrogen) according to manufacturer'sspecifications. Reverse transcription of the total RNA is performed in aRobocycler gradient 96 thermocycler (Stratagene) using the Advantage PCRkit according to the manufacturer's instructions (Clontech). Q-PCR isperformed using a 7500 Real Time PCR System (Applied Biosystems) andTaqMan® gene expression primer/probe sets (Applied Biosystems). Briefly,5 μl cDNA, 1.25 μl 20× Assays-on-Demand Gene Expression Assay Mix and12.5 μl 2× TaqMan® Universal PCR Master Mix in a total of 25 μl areamplified with the following thermocycler protocol: 1 cycle at 50° C.for 2 min; 1 cycle at 95° C. for 10 min; and 40 cycles at 95° C. for 15seconds/60° C. for 1 min. All data is analyzed with the SequenceDetection Software (version 1.2.3, Applied Biosystems), and expressionof each target gene normalized to an endogenous control gene. Eachexperiment is repeated twice and each sample is performed in triplicate.

Microarray Analysis

Microarray and bioinformatics analyses of the cells is performed usingthe U133A Human Genome Array from Affymetrix as a cooperative agreementwith Translational Genomics (TGen; Phoenix, Ariz.; Dr. Jeffrey Trent).

Comparative Genomic Hybridization Analysis: Genomic DNA Isolation

Genomic DNA is isolated from cells using the PUREGENE DNA isolation kit(Gentra Systems). Five μg of gDNA is digested with EcoRI, extracted withphenol:chloroform, ethanol precipitated, and resuspended in steriledistilled water, as previously described. O'Hagan et. al., 2003, CancerRes. 53:5352-5356.

Statistical Analysis

All statistical analyses are performed using Microsoft Excel'sspreadsheet software with the majority of statistics consisting of a“one-way analysis of variance” (ANOVA) determination with a value ofp<0.05 deemed significant. For the orthotopic mouse tumor formationstudies, we determined statistical significance using the Kruskal-WallisOne Way Analysis of Variance on Ranks, followed by Dunn's method or aone way analysis of variance (ANOVA) followed by theStudent-Newman-Keuls method for pairwise multiple comparisons. For theclonogenic and proliferation assays we determined statisticalsignificance using ANOVA followed by the Student-Newman-Keuls method forpairwise multiple comparisons. For the correlation of breast cancerstage and Nodal expression, a Spearman Rank Order Correlation wasemployed. In all cases, differences were statistically significant atP<0.05.

Analysis of DNA Methylation by Sequencing of Sodium Bisulfite-TreatedDNA

Genomic DNA is obtained by digestion with proteinase K (Quiagen)followed by phenol/chloroform extraction, and is subjected to sodiumbisulfite treatment to modify unmethylated cytosine to uracil using the‘CpGenome™ DNA Modification Kit’ (Chemicon International).Bisulfite-treated DNA is amplified by a nested-PCR protocol using theprimers described in Table 1.

TABLE 1 Primers used for amplifications after DNA bisulfate conversion.Primer Sequence NODAL gene CpG1 (52 CpGs) NODAL-1 EF5′-TTT TAG AAG GGA GTG AAT TGG-3′ (SEQ ID NO: 3) NODAL-1 ER5′-AAA AAA TAA AAA CTT CTA ATC TCC-3′ (SEQ ID NO: 4) NODAL-1 IF5′-AGT ATT TTA GTA AAT TTT TTA TTG-3′ (SEQ ID NO: 5) NODAL-1 IR5′-ATT AAT ATT ACT ATA ATA ATT TAA TC-3′ (SEQ ID NO: 6)NODAL gene CpG2 (47 CpGs) NODAL-2 EF5′-TAA TTT TAT AAG ATT GGA GAT TAG-3′ (SEQ ID NO: 7) NODAL-2 ER5′-TAC TAA AAC CCA AAA TAT AAA AAC-3′ (SEQ ID NO: 8) NODAL-2 IF5′-TTT AAA TTA AAA TTT AGA GAT AAT GG-3′ (SEQ ID NO: 9) NODAL-2 IR5′-ACT TTC AAA CCT AAC CAA CCC-3′ (SEQ ID NO: 10) LEFTY 1 (B) (61 CpGs)LEFTY1 EF 5′-TAG TTT TTA AGG TTT AGG GTG TG-3′ (SEQ ID NO: 11) LEFTY1 ER5′-TAC TAA CCC TAC TCT TAT CCC-3′ (SEQ ID NO: 12) LEFTY1 IF5′-AG TTT TAG TTG GGG TTT TTT AAG-3′ (SEQ ID NO: 13) LEFTY1 IR5′-TTA AAA ACC AAC ACA CAC CTA C-3′ (SEQ ID NO: 14)LEFTY 2 (A) (66 CpGs) LEFTY2 EF 5′-TAG TTT TTG AGG TTT AGG GTG TG-3′(SEQ ID NO: 15) LEFTY2 ER 5′-TAT CTC CTA ACC TAA CTA CC-3′(SEQ ID NO: 16) LEFTY2 IF 5′-AG TTT TAG TTG GGG TTT TTT AAG-3′(SEQ ID NO: 17) LEFTY2 IR 5′-CTC AAT AAC CCT ACC ATC CTC-3′(SEQ ID NO: 18) * EF/R = external primer set; IF/R = internal primer set

PCR is performed in a volume of 25 μl containing PCR Buffer (Qiagen);1.5 mM of MgCl2 (Qiagen); 200 μM of dNTPs (Invitrogen); 0.32 μM of eachprimer and 1 U of Hot Start Taq Plus DNA Polymerase (Qiagen). The PCRconditions are: 94° C. for 10 min, 94° C. for 3 min, 48° C. for 3 min,72° C. for 2 min one cycle; 94° C. for 3 min, 50° C. for 3 min, 72° C.for 2 min five cycles and 94° C. for 1 min, 52° C. for 1 min, 72° C. for1 min 35 cycles for the first reaction and the same annealingtemperatures (48°, 50° and 52° C.) for the nested reaction. Amplifiedproducts are purified using the Gel Purification Kit (Qiagen) and areligated to a vector using the TOPO TA Cloning Kit (Invitrogen). Twentyfour positive clones are sequenced for each sample using the vector'sforward and reverse primers. DNA sequencing reactions are performedusing the ‘DNA dRhodamine Terminator Cycle Sequencing Ready reaction’kit (Applied Biosystems) and an ABI3730x1 sequencer (Applied Biosystems)according to the manufacturer's instructions.

Immunoblotting

Protein lysates were prepared and quantified as previously described inHess et al., 2001, Cancer Res. 61:3250-3255. Equal amounts of proteinwere separated by SDS-polyacrylamide gel electrophoresis under reducingconditions, and the resolved proteins were transferred onto Immobilon-Pmembranes (Millipore Corp., Bedford, Mass.). Membranes were blocked in1% TBS, 0.1% Tween 20 (TBS-T) and 5% dry milk powder or 3% gelatin (forNodal Westerns). Blots were incubated with primary antibody (Table 2),washed in TBS-T or TBS-T containing 0.5M NaCl for the Nodal Westerns,and incubated with the appropriate horseradish peroxidase-labeledsecondary antibody. Secondary antibodies were detected by enhancedchemiluminescence (Super Signal; Pierce, Rockford, Ill.) and exposure toautoradiography film (Molecular Technologies, St Louis, Mo.). Nodalprotein was detected as two major bands at ˜48 and ˜35 kDa representingprecursor and pro-Nodal respectively. Nodal often appeared as multiplebands, likely due to degradation of protein modifications.

TABLE 2 Antibodies Utilized for Western Blot (WB), Immunohistochemical(IHC) and Immunofluorescence (IF) Analyses. Antibody Concentration & UseCompany Polyclonal goat  2 μg/mL, IF R&D Systems, anti-mNodal  2 μg/mL,IHC Minneapolis, MN Polyclonal rabbit 1:500, WB Santa CruzBiotechnology, anti-Nodal (H-110) Santa Cruz, CA Polyclonal goat 1:500,WB Santa Cruz Biotechnology, anti-Lefty 1:50, IF Santa Cruz, CAMonoclonal Mouse  1 μg/mL, WB R&D Systems, anti-Cripto 10 μg/mL, IFMinneapolis, MN Polyclonal Goat 1:20, IHC Santa Cruz Biotechnology,anti-Ki67 Santa Cruz, CA Monoclonal mouse 1:5000, WB ChemiconInternational, anti-Actin Temecula, CAImmunofluorescence

Cells were fixed with 4% paraformaldehyde, made permeable with 20 mMHepes, 0.5% TritonX-100 and blocked with serum-free protein block (DAKO,Carpinteria, Calif.). Primary antibodies were diluted in antibodydilutent (DAKO) to the concentrations outlined in SI Table 2, andappropriate fluorochrome-conjugated secondary antibodies were usedaccording to manufacturer recommendations. For certain images, nucleiwere stained with DAPI (0.1 mg/mL; Molecular Probes), and images wereobtained using confocal microscopy (Zeiss 510 META, Carl Zeiss Inc.).

Immunohistochemistry

Formalin-fixed, paraffin-embedded archival tissue was obtained frompatients with primary or metastatic cutaneous melanoma (LoyolaUniversity Chicago, Ill.). Immunohistochemical staining was performed ona HNS 710i Automated Immunostainer (Richard-Allan Scientific (RAS),Kalamazoo, Mich.) with the Multi-Species HRP/AEC Detection Systems.Following deparaffinization in xylene, ethanol degradation, and antigenretrieval with citrate buffer, four blocking steps were applied: 0.03%hydrogen peroxide, Avidin and Biotin blocks (Avidin/Biotin blocking kit,Vector Laboratories, Inc., Burlingame, Calif.), and a Serum-Free proteinblock. Anti-Nodal antibody (20 μg/mL, R&D Systems, Minneapolis, Minn.)was applied for 90 minutes. Slides were rinsed in TBS-T, incubated withbiotinylated anti-goat IgG (2 μg/ml, Vector Labs), washed with TBS-T andincubated with the streptavidin peroxidase reagent for 15 minutes. Colorwas produced with AEC (red) substrate (RAS) and counterstaining withMayer's hematoxylin. Samples were dehydrated in reagent grade alcoholand cover slipped with permanent mounting medium. Negative controlreactions were conducted with ChromPure Goat IgG (Jackson Labs), isotypematched and used at the same concentration as the Nodal antibody.Immunohistochemical staining for Nodal in a breast carcinoma progressionTMA (CBL-TMA-029; Creative Biolabs, Port Jefferson Station, N.Y.) wasperformed as previously described. Topczewska et al., 2006. Tissues fromthe orthotopic tumor models were formalin-fixed and paraffin-embeddedand immunohistochemical staining on this tissue was conducted using aKi67-specific antibody (Table 2) or ChromPure Goat IgG (Jackson Labs) aspreviously described. Topczewska et al., 2006. TUNEL assays to measureapoptosis were conducted as per instructions (Upstate).

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims. The combination ofparticular aspects of the various embodiments of the invention isincluded in the scope of the invention. All patents, patentapplications, and other scientific or technical writings referred toanywhere herein are incorporated by reference in their entirety.

What is claimed is:
 1. A method of detecting aggressive metastaticmelanoma or metastatic breast carcinoma tumor cells in a human patientcomprising the steps of: a. obtaining a sample of tumor cells from thehuman patient, wherein said sample presents Nodal in patients withaggressive metastatic melanoma or metastatic breast carcinoma tumorcells; b. assaying the sample for the presence of Nodal protein andLefty protein; and c. detecting aggressive metastatic melanoma ormetastatic breast carcinoma tumor cells if Nodal protein is present andLefty protein is absent in the sample.
 2. The method of claim 1, whereinassaying for the presence of Nodal and the absence of Lefty comprises aprotein based assay.